Therapeutic agents comprising pro-apoptotic proteins

ABSTRACT

The present invention relates to targeted killing of a cell utilizing a chimeric polypeptide comprising a cell-specific targeting moiety and a signal transduction pathway factor. In a preferred embodiment, the signal transduction pathway factor is an apoptosis-inducing factor, such as granzyme B, granzyme A, or Bax.

[0001] The present invention claims priority to U.S. Provisional PatentApplication Serial No. 60/306,091, filed Jul. 17, 2001; to U.S.Provisional Patent Application Serial No. 60/332,886, filed Nov. 6,2001; and to U.S. Provisional Patent Application Serial No. 60/360,361,filed Feb. 28, 2002, all of which are incorporated by reference hereinin their entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to the fields of cellular andmolecular biology and cancer biology. More particularly, the presentinvention provides methods and compositions concerning therapeuticagents comprising a pro-apoptosis moiety and a cell-specific targetingmoiety.

BACKGROUND OF THE INVENTION

[0003] The selective destruction of an individual cell is oftendesirable in a variety of clinical settings. A multitude of signaltransduction pathways in the cell are linked to its death and survival,and delivery of a limiting and/or crucial component of the pathway canbe productive in terms of its destruction. A classic example of such asignal transduction pathway is apoptosis, and a variety of elements ofapoptotic pathways would be useful to target a cell for death.Apoptosis, or programmed cell death, is a fundamental processcontrolling normal tissue homeostasis by regulating a balance betweencell proliferation and death (Vaux et al., 1994; Jacobson et al., 1997).

[0004] The serine protease granzyme B (GrB) (Lobe et al., 1986; Schmidand Weissman, 1987; Trapani et al., 1988) is integrally involved inapoptotic cell death induced in target cells upon their exposure to thecontents of lysosome-like cytoplasmic granules (or cytolytic granules)found in cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells(Henkart, 1985; Young and Cohn, 1986; Smyth and Trapani, 1995).Cytotoxic lymphocyte granules contain perforin, a pore-forming protein,and a family of serine proteases, termed granzymes (Table 1). Perforinhas some structural and functional resemblance to the complementproteins C6, C7, C8 and C9, members of complement membrane attackcomplex (Shinkai et al., 1988). In lymphocyte-mediated cytolysis,perforin is inserted into the target cell membranes and appears topolymerize to form pores (Podack, 1992; Yagita et al., 1992), whichmediates access of granzyme B to the target cell cytoplasm. Once inside,granzyme B induces apoptosis by directly activating caspases andinducing rapid DNA fragmentation (Shi et al., 1992). TABLE 1 GRANZYMES(LYMPHOCYTE SERINE PROTEASES) Enzyme Names Species Other Names ActivityA Mouse Hanukah factor, MTSP, SE-1, CTLA-3 Tryptase Rat RNKP-2,fragmentin 1 Human Hanukah factor, HTSP-1, granzyme 1 B Mouse CCP-1,CTLA-1 Asp-ase Rat Fragmentin 2, RNKP-1 Human HLP, granzyme 2, HSE26.1,CSPB C Mouse CCP-2 Unknown Rat RNKP-4 D Mouse CCP-5 Unknown E MouseCCP-3, MCSP2 Unknown F Mouse CCP-4, MCSP3 Unknown G Mouse MCSP1 UnknownH Human CCP-X, CSP-C Chymase I Rat GLP I and II Unknown J Rat RNKP-5Unknown K Rat Tryptase 2, fragmentin 3 Tryptase Human Granzyme 3Tryptase M Rat RNK-Met-1 Met-ase Human Met-ase

[0005] The granzymes are structurally related, but have diversesubstrate preference. Through its unique ability to cleave afteraspartate residues, granzyme B can cleave many procaspases in vitro, andit has been an important tool in analyzing the maturation of caspase-3(Darmon et al., 1995; Quan et al., 1996; Martin et al., 1996), caspase-7(Chinnaiyan et al., 1996; Gu et al., 1996; Femandes-Alnemri et al.,1995), caspase-6 (Orth et al., 1996; Femandes-Alnemri et al., 1995),caspase-8 (Muzio et al., 1996), caspase-9 (Duan et al., 1996), andcaspase-10a/b (Fernandes-Alnemri et al., 1996; Vincenz and Dixit, 1997).Furthermore, it is highly toxic to target cells (Shi et al., 1992). Ithas been assumed until now that granzyme B kills cells by direct caspaseactivated, supplemented under certain circumstances by direct damage todownstream caspase substrates (Andrade et al., 1998). Having gainedaccess to the cytosol, granzyme B is rapidly translocated to the nucleus(Jans et al., 1996; Trapani et al., 1996) and can cleave poly(ADP-ribose) polymerase and nuclear matrix antigen, sometimes usingdifferent cleavage sites than those preferred by caspases (Andrade etal., 1998). Although many procaspases are efficiently cleaved in vitro,granzyme B-induced caspase activation occurs in a hierarchical manner inintact cells, commencing at the level of executioner caspases such ascaspase-3, followed by caspase-7 (Yang et al., 1998). This is incontrast to FasL-mediated killing, which relies on a membrane signalgenerated through apical caspases such as caspase-8 (Muzio et al., 1996;Sarin et al., 1997). In addition, some studies showed that granzyme Bcan also induce death through a caspase-independent mechanism thatinvolves direct damage to nonnuclear structures, although the keysubstrates in this pathway have yet to be elucidated (Sarin et al.,1997; Trapani et al., 1998; Heibein et al., 1999; Beresford et al.,1999).

[0006] Studies by Froelich and co-workers suggest that GrB isinternalized by receptor-mediated endocytosis, and that the role ofperforin is to mediate release of granzyme B from endocytic vesicles. Infact, perforin can be replaced by other vesicle-disrupting factors suchas those produced by adenovirus (Froelich et al., 1996; Pinkoski et al.,1998; Browne et al., 1999).

[0007] Granzymes in general are highly homologous, with 38-67% homologyto GrB (Haddad et al., 1991), and they contain the catalytic triad(His-57, Asp-102, and Ser-195) of trypsin family serine proteases. Otherfeatures include the mature, N-terminal Ile-Ile-Gly-Gly sequence, threeor four disulfide bridges, and a conserved motif (PHSRPYMA), which alsoappears in neutrophil cathepsin G and mast cell chymases. Thecarbohydrate moieties of granzymes are Asn-linked (Griffiths and Isaaz,1993). The granzyme mRNA transcripts are translated aspre-pro-proteases. The pre- or leader sequence is cleaved by signalpeptidase at the endoplasmic reticulum. When the propeptides areremoved, the inactive progranzymes (zymogens) become active proteases.The granzyme propeptides sequences start after the leader peptide andend before the N-terminal Ile needed for the protease to fold into acatalytic conformation (Kam et al., 2000).

[0008] Among the various apoptotic factors identified so far, members ofthe Bcl-2 family represent some of the most well-defined regulators ofthis death pathway. Some members of the Bcl-2 family, including Bcl-2,Bcl-XL, Ced-9, Bcl-w and so forth, promote cell survival, while othermembers including Bax, Bcl-Xs, Bad, Bak, Bid, Bik and Bim have beenshown to potentiate apoptosis (Adams and Cory, 1998). A number ofdiverse hypotheses have been proposed so far regarding the possiblebiological functions of the Bcl-2 family members. These include dimerformation (Oltvai et al., 1993), protease activation (Chinnaiyan et al.,1996), mitochondrial membrane depolarization (6), generation of reactiveoxygen intermediates (Hockenbery et al., 1993), regulation of calciumflux (Lam et al., 1994; Huiling et al., 1997), and pore formation(Antonsson et al., 1997; Marzo et al., 1998).

[0009] Bax, a 21 kDa death-promoting member of the Bcl-2 family, wasfirst identified as a protein that co-immunoprecipated with Bc1-2 fromdifferent cell lines (Oltvai et al., 1993). Overexpression of Baxaccelerates cell death in response to a wide range of cytotoxic results.Determination of the amino acid sequence of the Bax protein showed it tobe highly homologous to Bcl-2. The Bax gene consists of six exons andproduces alternative transcripts, the predominant form of which encodesa 1.0 kb mRNA and is designated Baxα. Like Bcl-2 and several othermembers of the Bcl-2 family, the Bax protein has highly conservedregions, BH1, BH2 and BH3 domains, and hydropathy analysis of thesequences of these proteins indicates the presence of a hydrophobictransmembrane segment at their C-terminal ends (Oltvai et al., 1993).

[0010] Bax is widely expressed without any apparent tissue specificity.However, on the induction of apoptosis, Bax translocates intomitochondria, resulting in mitochondria dysfinction and release ofcytochrome c, which subsequently activates caspase pathways (Hsu andYoule, 1997; Wolter et al., 1997; Gross et al., 1998). Thistranslocation process is rapid and occurs at an early stage of apoptosis(Wolter et al., 1997). Selective overexpression of Bax in human ovariancancer through adenoviral gene transfer resulted in significant tumorcell kill in vivo (Tai et al., 1999). Overexpression of the Bax gene bya binary adenovirus system in cultured cell lines from human lungcarcinoma results in caspase activation, apoptosis induction, and cellgrowth suppression. Moreover, intratumoral injection of adenovirusvector expressing the Bax gene suppressed growth of human lung cancerxenografts established in nude mice (Kagawa et al., 2000; Kagawa et al.,2000).

[0011] WO 99/45128 and Aqeilan et al. (1999) are directed to chimericproteins having cell-targeting specificity and apoptosis-inducingactivities, particularly the recombinant chimeric protein IL-2-Bax,which specifically targets IL2 receptor-expressing cells and inducescell-specific apoptosis.

[0012] WO 99/49059 relates to a chimeric toxin comprised of gonadotropinreleasing hormone (GnRH) and Pseudomonas exotoxin A (PE) to detect atumor-associated epitope expressed by human adenocarcinoma.

[0013] WO 97/46259 concerns targeted chimeric toxins comprising celltargeting moieties and cell killing moieties directed to neoplasticcells. In a specific example, the chimeric toxin comprises gonadotropinreleasing hormone homologs and Pseudomonas Exotoxin A.

[0014] WO 97/22364 addresses targeted treatment of allergy responses,whereby a chimeric cytotoxin Fc_(2′-3)-PE₄₀ is directed to targetedelimination of cells expressing the FcεRI receptor.

[0015] While some chimeric protein compositions have been described,other methods and compositions are needed for improved therapiesinvolving the killing of cells.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to methods and compositionsinvolving the delivery of chimeric polypeptides comprising signaltransduction pathway factors that induce death of a targeted cell. In apreferred embodiment, this factor is a pro-apoptotic factor.

[0017] Almost all cells contain mechanisms responsible for mediatingcell-death (apoptosis). Thus, in some embodiments, the present inventionaddresses delivery of certain pro-apoptotic proteins that are centralmediators of this effect to the interior of target cells, which willresult in cell death through apoptotic mechanisms. Theapoptosis-inducing moiety induces programmed cell death upon entry intothe target cell of the chimeric polypeptide, which is delivered forbinding to the target cell by the cell-specific targeting moiety. Insome embodiments of the present invention and as an advantage over knownmethods in the art, pro-apoptotic polypeptides are delivered as proteinsand not as nucleic acid molecules to be translated to produce thedesired polypeptides. As an additional advantage, human sequences areutilized in the chimeric polypeptides of the present invention tocircumvent any undesirable immune responses from a foreign polypeptide.

[0018] In further embodiments, granzyme A or granzyme B is a mediatorfor inducing apoptosis. In specific embodiments, recombinant ligand(VEGF) and/or recombinant antibody (scFvMEL) moieties are fused asnucleic acid sequences to those sequences that encode a granzyme or aBcl-2 family member. The inventors present data herein demonstratingthat chimeric polypeptides, such as granzymeB-vegf121 andgranzymeB-scFvMEL, are cytotoxic to target cells. Given that a skilledartisan recognizes that there are multiple similar cell-targeting andpro-apoptotic examples that may be used interchangeably with thespecific examples herein, this indicates that constructs containingpro-apoptotic proteins have significant therapeutic potential for thetreatment of disease states and represent a new class of therapeuticagents with a novel mechanism of action.

[0019] In an embodiment in which pro-apoptotic proteins are utilized asthe killing moiety in chimeric proteins, recombinant antibody (scFvMEL)that binds to the cell-surface antigen gp240 of melanoma cells and isinternalized efficiently is utilized. The inventors fused the genesencoding scFvMEL to genes encoding Bax, truncated Baxl-5, and Bax 345,respectively (designated as scFvMEL-bax, scFvMEL-Baxl-5 and scFvMEL-Bax345, respectively). These genes were inserted into protein-expressionvectors and transformed into bacteria. The fusion proteins werepurified, tested against target cells in culture, and shown to becytotoxic to target cells. This suggests that constructs containing thepro-apoptotic protein Bax have significant therapeutic potential for thetreatment of diseases and present a new class of therapeutic agents witha novel mechanism of action.

[0020] In an object of the present invention, there is a chimericpolypeptide comprising a cell-specific targeting moiety and a signaltransduction pathway factor.

[0021] In another object of the present invention, there is a chimericpolypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme. In a specific embodiment, the granzyme is granzyme B. Inanother specific embodiment, the amino acid sequence of said granzyme Bis selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In anotherspecific embodiment, the amino acid sequence of said granzyme B is SEQID NO:60, SEQ ID NO:60 further comprising an N-terminal extension of SEQID NO:61, or SEQ ID NO:60 wherein the first twenty amino acids areabsent. In a further specific embodiment, the amino acid sequence ofsaid granzyme B is at least 100 contiguous amino acids from SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, or SEQ ID NO:60. In a further specific embodiment, the amino acidsequence of said granzyme B is at least 75 contiguous amino acids fromSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, or SEQ ID NO:60. In a further specific embodiment, theamino acid sequence of said granzyme B is at least 40 contiguous aminoacids from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, or SEQ ID NO:60. In an additional specificembodiment, the granzyme is granzyme A. In a further specificembodiment, the amino acid sequence of said granzyme A is selected fromthe group consisting of SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. Ina further specific embodiment, the amino acid sequence of said granzymeA is at least 100 contiguous amino acids from SEQ ID NO:23, SEQ ID NO:24or SEQ ID NO:25. In a further specific embodiment, the amino acidsequence of said granzyme A is at least 75 contiguous amino acids fromSEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. In a further specificembodiment, the amino acid sequence of said granzyme A is at least 40contiguous amino acids from SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.In an additional specific embodiment, the cell-specific targeting moietyis a cytokine, an antibody, a ligand, or a hormone. In a furtherspecific embodiment, the ligand is VEGF. In a further specificembodiment, the VEGF is vegf121. In a further specific embodiment, theantibody is a single chain antibody. In a further specific embodiment,the single chain antibody is scFvMEL. In an additional specificembodiment, the granzyme is granzyme B and said cell-specific targetingmoiety is vegf121. In another specific embodiment, the granzyme isgranzyme B and said cell-specific targeting moiety is scFvMEL. In afurther specific embodiment, the polypeptide further comprises a linker,such as SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. In a specificembodiment, the polypeptide is encoded by a recombinant polynucleotide.

[0022] In an additional object of the present invention, there is anexpression cassette comprising a polynucleotide encoding a chimericpolypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme, and wherein said polynucleotide is under control of aregulatory sequence operable in a host cell. In specific embodiments,the granzyme is granzyme A or granzyme B. In a specific embodiment, thegranzyme A is encoded by a polynucleotide of SEQ ID NO:26, SEQ ID NO:27,or SEQ ID NO:28. In another specific embodiment, the granzyme B isencoded by a polynucleotide of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. In an additional specificembodiment, the cassette is comprised in a recombinant viral vector,such as an adenoviral vector, an adeno-associated viral vector, or aretroviral vector.

[0023] In an additional object of the present invention, there is a hostcell comprising an expression cassette comprising a polynucleotideencoding a chimeric polypeptide comprising a cell-specific targetingmoiety and an apoptosis-inducing factor, wherein said apoptosis-inducingfactor is a granzyme. In specific embodiments, the cell is furtherdefined as a prokaryotic host cell or an eukaryotic host cell.

[0024] In another object of the present invention, there is a method ofusing a host cell comprising an expression cassette comprising apolynucleotide encoding a chimeric polypeptide comprising acell-specific targeting moiety and an apoptosis-inducing factor, whereinsaid apoptosis-inducing factor is a granzyme, comprising culturing thehost cell under conditions suitable for the expression of the chimericpolypeptide.

[0025] In an additional object of the present invention, there is amethod of inducing apoptosis in a cell, comprising administering to saidcell an effective amount of a chimeric polypeptide comprising acell-specific targeting moiety and a granzyme. In specific embodiments,the granzyme is granzyme A or granzyme B. In specific embodiments, thecell is in vivo and/or in a human.

[0026] In another object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a granzyme, wherein said cell-specific targetingmoiety is scFvMEL and said granzyme is granzyme B. It is contemplatedthat the cell-specific targeting moiety acts by targeting specificcells, for example, cells that express on their surface a peptide orpolypeptide that is capable of specifically binding the targetingmoiety. The compound that allows the cell to be specifically targetedmay be referred herein as the target. Thus, in some embodiments of theinvention, cells may have a target to which the cell-specific targetingmoiety recognizes.

[0027] In another object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a granzyme, wherein said cell-specific targetingmoiety is vegf121 and said granzyme is granzyme B.

[0028] In an additional object of the present invention, there is amethod of inducing apoptosis in a cell, comprising administering to saidcell an effective amount of a chimeric polypeptide comprising acell-specific targeting moiety and a pro-apoptotic member of the Bcl-2family. In a specific embodiment, the pro-apoptotic member of the Bcl-2family is Bax or a fragment thereof. In specific embodiments, the cellis in vivo and/or in a human. In a specific embodiment, the fragment ofBax lacks at least part of a polypeptide encoded by exon 6 in a Baxpolynucleotide sequence.

[0029] In another object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a pro-apoptotic member of the Bcl-2 family, whereinsaid cell-specific targeting moiety is scFvMEL and said pro-apoptoticmember of the Bcl-2 family is Bax or a fragment of Bax. In a specificembodiment, the fragment of Bax lacks at least part of exon 6 in a Baxpolynucleotide sequence.

[0030] In an additional object of the present invention, there is amethod of treating a disease in an individual, comprising the steps ofadministering to said individual a therapeutically effective amount of acomposition comprising a chimeric polypeptide comprising anapoptosis-inducing moiety and a cell-specific targeting moiety; and apharmaceutical carrier. In a specific embodiment, the pharmaceuticalcarrier comprises a lipid. In another specific embodiment, the diseaseis cancer, diabetes, arthritis, or inflammatory bowel disease,atherosclerosis, or diabetic retinopathy. In an additional specificembodiment, the disease is cancer. In a further specific embodiment, theapoptosis-inducing moiety is a granzyme. In a further specificembodiment, the granzyme is granzyme B or a fragment thereof. In anadditional specific embodiment, the apoptosis-inducing moiety is apro-apoptotic member of the Bcl-2 family. In another specificembodiment, the pro-apoptotic member of the Bcl-2 family is Bax or afragment thereof. In an additional specific embodiment, the fragment ofBax lacks at least part of a polypeptide encoded by exon 6 in a Baxpolynucleotide sequence. In another specific embodiment, the fragment ofBax lacks at least part of a polypeptide encoded by exons selected fromthe group consisting of 4, 5, and 6. In an additional specificembodiment, the administration is by intravenous injection. In anotherspecific embodiment, the administration is by inhalation. In a furtherspecific embodiment, the administration is intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally, byinhalation (e.g. aerosol inhalation), by injection, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in a creme, or in a lipidcomposition. In a specific embodiment, the method further comprisesadministering to said individual an anti-inflammatory composition,chemotherapy, surgery, radiation, hormone therapy, or gene therapy.

[0031] It is contemplated that aspects of the invention discussed in thecontext of one embodiment of the invention may be employed with respectto any other embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0032] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0033]FIG. 1 illustrates human pre-mature granzyme B cDNA from Hut78cells. A 1% agarose gel electrophoresis demonstrates human pre-maturegranzyme B cDNA synthesized from Hut78 cells by RT-PCR. Lane 1represents a low mass DNA molecular marker; lane 2 represents controlsynthesized CDNA (˜500 bp); lane 3 represents no RT control; and lane 4represents human pre-mature granzyme B CDNA (˜800 bp).

[0034]FIG. 2 shows the nucleotide sequence encoding human pre-maturegranzyme B (SEQ ID NO:54) and amino acid sequence (SEQ ID NO:55).

[0035]FIG. 3 illustrates construction of pET32GrB-vegf121 andpET32GrB-scFvMEL fusion constructs. Construction of these fusionconstructs was based on a PCR method. FIG. 3A shows the construction ofpET32GrB-vegf121. FIG. 3B shows the construction of pET32GrB-scFvMEL.The full length genes were ligated into the Xba I/Xho I site of theexpression vector pET-32a(+).

[0036]FIG. 4 demonstrates the predicted structure of recombinantgranzyme B-vegf121 (FIG. 4A), granzyme B-scFvMEL (FIG. 4B) in pET32avector expressed in E. coli and the sequences of granzyme B-vegf121(FIG. 4C and 4D) (SEQ ID NO:56 for nucleic acid sequence and SEQ IDNO:57 for amino acid sequence), granzyme B-scFvMEL (FIG. 4E and 4F) (SEQID NO:58 for nucleic acid sequence and SEQ ID NO:59 for amino acidsequence). The pET32a(+) vector contains a T7 promoter for high-levelexpression. Expression of the nucleic acid includes sequence containingthe Trx.tag, followed by a His.tag, a thrombin cleavage site, and anenterokinase cleavage site for final removal of the protein purificationtag.

[0037]FIG. 5 shows SDS-PAGE analysis of the expression of the fusionproteins SDS-PAGE Coomassie Blue staining of granzyme B-vegf121 (FIG.5A) and granzyme B-scFvMEL (FIG. 5B) under reducing conditions. Panel Aof FIG. 5A shows SDS-PAGE Coomassie Blue staining of Granzyme B-vegf121.Lane 1 shows non-induced total cell lysates; lane 2 shows induced totalcell lysates; lane 3 shows non-induced soluble; lane 4 shows inducedsoluble; lane 5 shows non-induced insoluble; lane 6 shows inducedinsoluble; lane 7 shows protein molecular marker. In Panel B, lane 1shows protein molecular marker; lane 2 shows pro-granzyme B-vegf121(IMAC-eluate from Talon Resin); lane 3 shows pro-granzyme B-vegf121(IMAC-Elute from Nickel NTA), Lane 4: Granzyme B-vegf121 (after rEKcut). In FIG. 5B, there is shown SDS-PAGE Coomassie blue staining ofgranzyme B-scFvMEL. In Panel C, lane 1 shows protein molecular marker;lane 2 shows non-induced total cell lysates; lane 3 shows induced totalcell lysates; lane 4 shows non-induced soluble; lane 5 shows inducedsoluble; lane 6 shows non-induced insoluble; lane 7 shows inducedinsoluble. In Panel D, lane 1 shows protein molecular marker; lane 2shows pro-granzyme B-scFvMEL (IMAC-eluate from Nickel NTA); lane 3 showsgranzyme B-scFvMEL (after rEK cut).

[0038]FIG. 6 demonstrates a Western blot analysis of granzyme B-vegf121and granzyme B-scFvMEL.

[0039]FIG. 7 shows binding activity of scFvMEL moiety of granzymeB-scFvMEL fusion protein. ELISA of different scFvMEL fusion proteinswere examined on a plate pre-coated with Protein L.

[0040]FIG. 8 demonstrates testing of cytotoxicity of granzyme B-vegf121against log-phase PAE-Flk-1 and PAE-Flt-1

[0041]FIG. 9 demonstrates testing of cytotoxicity of granzyme B-scFvMELon A375-M.

[0042]FIG. 10 illustrates the human Bax gene, its exons, and domainsBH1, BH2, and BH3.

[0043]FIG. 11 demonstrates cloning of human Bax cDNA from Namalwa cellsby PCR. Lane 1:Low Mass DNA Molecular Marker, lanes 2-6: Controlsynthesized cDNA (˜500 bp), lanes 7-8:Human Bax cDNA (˜580 bp) usingrandom primer (lane 7) and using Oligo(dT) primer (lane 8).

[0044]FIGS. 12A and 12B illustrate construction of scFvMEL-bax-relatedfusion constructs.

[0045]FIG. 13 shows SDS-PAGE and Coomassie Blue Staining analysis of theexpression of the fusion proteins.

[0046]FIG. 14 shows the expression of pET32-scFvMEL-bax andpET32-Bax-scFvMEL transformed into AD494(DE3)pLysS E coli and under IPTGinduction.

[0047]FIG. 15 demonstrates western blotting analysis of the expressionof the full length bax and Bax-scFvMEL proteins. Lane 1:pBad/HisA(negative control), Lane 2:pBad/HisLacZ (expression positive control),Lane 3-5:Bax protein (lane 3:expression in RM+glucose+ampicillin, lane4:expression in RM+ampicillin, lane 5:expression in LB+ampicillin),Lanes 6-8:Bax-scFvMEL protein (lane6:expression in RM+glucose+ampicillin, lane 7:expression in RM+ampicillin, lane8:expression in LB+ampicillin).

[0048]FIGS. 16A and 16B demonstrate the binding activity of scFvMELmoiety of fusion proteins.

[0049]FIG. 17 shows the cytotoxicity of scFvMEL-bax345 andBax345-scFvMEL fusion proteins on A375-M.

[0050]FIG. 18 shows ELISA of granzyme B-Vegf121 on various cell lines(detected with mouse anti-vegf121 antibody and mouse anti-granzyme Bantibody).

[0051]FIG. 19 demonstrates cytotoxicity of Granzyme B-VEGF121 ontransfected endothelial cells.

[0052]FIG. 20 shows cytotoxicity assay of granzyme B-Vegf121 vs. vegf121rgel in vitro against PAE/FLK-1.

[0053]FIG. 21 illustrates caspase activity on PAE cells treated withGranzyme B-Vegf121.

[0054]FIG. 22 demonstrates cytochrome c release of PAE cells treatedwith GRB/VEGF121.

[0055]FIG. 23 shows Bax translocation of PAE cells after GRB/VEGF121treatment.

[0056]FIG. 24 illustrates cytochrome c release in A375-M vs. SKBR3-HPcells treated with GRB/scFvMEL.

[0057]FIG. 25 illustrates GrB/VEGF121 induces DNA laddering on PAE/flk-1cells.

[0058]FIG. 26 shows ELISA of GrB/scFvMEL on gp240 Ag-positive A375-M vsgp240 Ag-negative T-24 cells detected by Grb mouse mAb.

DETAILED DESCRIPTION OF THE INVENTION

[0059] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

[0060] As used herein the specification, “a” or “an” may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising”, the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

[0061] The term “apoptosis” as used herein is defined as programmed celldeath; an endogenous cell death program results in the death of thecell.

[0062] The term “cytokine” as used herein is defined as an agent made bya cell that affects the behavior of another cell. In a specificembodiment, the agent is a polypeptide. For example, cytokines made bylymphocytes are often called lymphokines or interleukins (IL).Furthermore, cytokines act on specific cytokine receptors on the cellsthat they affect. In a specific embodiment, the term “cytokine” includesgrowth factors.

[0063] The term “granzyme” as used herein is defined as an enzyme fromthe granules of cytotoxic lymphocytes that, upon entry into the cytosolof a cell, induce apoptosis and/or nuclear DNA fragmentation. In aspecific embodiment, the granzyme is a lymphocyte serine protease. Insome embodiments, the granzyme is full-length, whereas in otherembodiments the granzyme is partial.

[0064] The term “signal transduction pathway factor” as used herein isdefined as an enzyme, substrate, cofactor or other protein whichinfluences biological activity of another enzyme, cofactor or protein.In a specific embodiment, the factor is associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a growth response within the cell. In oneembodiment, the growth response that is modulated is a pro-growthresponse. In an alternative embodiment, the growth response that ismodulated is an anti-growth response, such as induction of apoptosis.

[0065] The present invention relates to chimeric proteins withcell-targeting specificity and cell-destruction moieties, such as fromsignal transduction pathways linked, either directly or indirectly, tocell death. In some embodiments, the cell-destruction moieties areapoptosis-inducing activities. The chimeric proteins of the inventionare composed of a cell-specific targeting moiety and anapoptosis-inducing moiety. The cell-specific targeting moiety providescell-specific binding properties to the chimeric protein, while theapoptosis-inducing moiety induces programmed cell death upon entry intoa target cell. In some embodiments, the chimeric proteins of theinvention are delivered as polypeptides and are produced by recombinantexpression of a fusion polynucleotide between a coding sequence of acell-targeting moiety and a coding sequence of an apoptosis-inducingprotein. Such chimeric proteins are likely to be superior to theimmunotoxins currently used in the art because they are of human originand thus are expected to have reduced immunogenicity in a humanrecipient. In addition, chimeric proteins kill target cells by inducingapoptosis which does not cause a release of cellular organelles into theextracellular environment to result in an inflammatory response. Whencells die by the apoptotic pathway, they shrink and condense, but theorganelles and plasma membranes retain their integrity, and the deadcells are rapidly phagocytosed by neighboring cells or macrophagesbefore there is leakage of the cells' contents, thereby elicitingminimal tissue or systemic response.

[0066] The invention also relates to pharmaceutical compositions of thechimeric proteins, methods of producing such proteins and methods ofusing the same in vitro and in vivo, especially for eliminating specificundesirable target cells, and for the treatment of a variety of diseaseconditions as well as the use of the proteins for disease diagnosis.

[0067] In the present invention, methods and compositions regardingtargeted destruction of a cell utilizing a chimeric polypeptide aredisclosed. The chimeric polypeptide is comprised of at least twomoieties: one moiety is the effectual component for killing of the cell;the second moiety is the delivery component of the chimeric polypeptideto target the killing component to the cell of interest. In someembodiments of the present invention, at least one of the moieties, andpreferably both, are of human origin, which eliminates an immuneresponse from the individual to whom the chimeric polypeptide isadministered. In one embodiment, the moiety for killing the cell is acomponent of a signal transduction pathway, such as one which is alimiting factor or restriction point in the pathway. Delivery of thesignal transduction pathway bypasses the requirement to elicit upstreamsteps of the pathway, and the resultant administration of thisrestriction point ends in the same effect, which is destruction of thecell. A skilled artisan recognizes that types of agents which could bedelivered intracellularly to mediate signal transduction includeenzymes, such as kinases (for example, protein kinase B (PKB,AKT), whichmediates insulin signaling; protein kinase C, which is involved innumerous signaling events; and phosphatidylinositol 3-kinase, which isinvolved in numerous signaling events); phosphatases; proteases (such ascaspase 3); nucleases (such as caspase-activated deoxyribonuclease(CAD), which is a mediator of apoptosis); phospholipases; NCKAP1 (whichis an apoptosis-related protein downregulated in the brain tissues ofAlzheimer's patients; Suzuki et al., 2000) or co-factors, such ascytochrome c (which is involved in apoptosis signaling) and cyclic AMP(which is involved in numerous pathways).

[0068] In a specific embodiment, the signal transduction pathway factoris an enzyme. The enzyme may be a hydrolase (e.g., deaminase, esterase,glycosidase, lipase, nuclease, peptidase, phosphatase,phosphodiesterase, and proteinase); isomerase (e.g., epimerase, mutase,and racemase); ligase or synthetase (e.g., acyl-CoA synthetase,amino-acyl-tRNA synthetase, and carboxylase); lyase (e.g., aldolase,decarboxylase, dehydratase, and nucleotide cyclase); oxidoreductase(e.g., dehydrogenase, dioxygenase, hydrogenase, monooxygenase,nitrogenase, oxidase, and reductase); and/or transferase (e.g.,acyltransferase, aminotransferase, glycosyltransferase, kinase,methyltransferase, nucleotidyltransferase, phosphorylase, andsulphotransferase). In specific embodiments, the enzyme is classified asa toxin, which means it is toxic to a cell, tissue, or organism.

[0069] In some embodiments, the signal transduction pathway factor is anapoptosis-inducing factor. Almost all cells contain mechanismsresponsible for mediating cell death (apoptosis). In a specificembodiment, and as demonstrated in the Examples herein, delivery ofgranzyme B protein into the interior of target cells results in celldeath through apoptotic mechanisms. Using recombinant ligand (VEGF) andrecombinant antibody (scFvMEL), which bind to the cell-surface of tumorcells and internalize efficiently, the inventors designed two novelgranzyme B-related fusion proteins: GrB-vegf121 to specifically targetthe endothelial cells; and GrB-scFvMEL to specifically target themelanoma cells.

[0070] A skilled artisan recognizes particular cell-specific targetingmoieties which would be useful in the chimeric polypeptide to target acell of interest. For example, the cell-specific targeting moieties maybe antibodies to a particular cell marker(s), growth factor(s),hormone(s), or cytokine(s).

[0071] A skilled artisan is aware that nucleic acid sequences and aminoacid sequences useful for generating the chimeric polypeptide of thepresent invention are readily obtainable, particularly through publicdatabases, such as the National Center for Biotechnology Information's(NCBI) GenBank database, or commercially available databases such asfrom Celera Genomics, Inc. (Rockville, Md.). For example, granzyme Bamino acid sequences useful in the present invention may include,followed by their GenBank Accession number, at least: P10144 (SEQ IDNO:11); XP_(—)012328 (SEQ ID NO:12); A61021 (SEQ ID NO:13); NP_(—)004122(SEQ ID NO:14); CAA01810 (SEQ ID NO:15); and/or AAA75490 (SEQ ID NO:16).SEQ ID NO:60 is human granzyme B sequence reflecting variances seen inSEQ ID NO:11 through SEQ ID NO:16, such as at residue 55 (a Gln or anArg), at residue 94 (a Pro or an Ala), as an N-terminal extensioncomprising SEQ ID NO:61 (MKSLSLLHLFPLPRAKREQGGNNSSSNQGSLPEK), and/or asa deletion of residues 1 through 20.

[0072] Granzyme B nucleic acid sequences useful in the present inventionmay include at least: XM_(—)012328 (SEQ ID NO:17); BF589964 (SEQ IDNO:18); BF221604 (SEQ ID NO:19); NM_(—)004131 (SEQ ID NO:20); A26437(SEQ ID NO:21); and/or M28879 (SEQ ID NO:22). Granzyme A amino acidsequences useful in the present invention may include, followed by theirGenBank Accession number, at least: P12544 or NP_(—)006135 (SEQ IDNO:23) or XP_(—)003652 (SEQ ID NO:24). SEQ ID NO:25 comprises a humangranzyme A amino acid sequence and reflects variance in SEQ ID NO:23 andSEQ ID NO:24 at residue 121 (Thr or Met, respectively). Granzyme Anucleic acid sequences useful in the present invention may include atleast: XM_(—)003652 (SEQ ID NO:26); NM_(—)006144 (SEQ ID NO:27); and/orU40006 (SEQ ID NO:28). A skilled artisan recognizes how to retrievethese and related sequences from the NCBI GenBank database.

[0073] I. Apoptosis-Inducing Proteins

[0074] Strictly regulated cell death is required for the development ofmultilineage organisms and the maintenance of homeostasis withintissues. Differentiation status of an individual cell directly affectswhether it can execute a suicidal response following a death stimulusvaries. Both positive and negative regulators of programmed cell death(apoptosis) have been identified. Bcl-2 is a repressor of programmedcell death (Vaux et al., 1988), and recently, other Bcl2 homologues wereshown to inhibit apoptosis. However, one homolog of Bcl-2, Bax, mediatesan opposite effect through acceleration of apoptosis. In the Bcl-2family there is notable homology clustered within two conserved regions:Bcl-2 homology domains 1 and 2 (BH1 and BH2) (Oltvai et al., 1993; Boiseet al., 1993; Kozopas et al., 1993; Lin et al., 1993). Members of theBcl family include Bax, Bcl-X_(L), Mcl-1, Al and several open readingframes in DNA viruses. Another conserved domain in Bax, distinct fromBH1 and BH2, is termed BH3 and mediates cell death and protein bindingfunctions (Chittenden et al., 1995). A subset of the pro-apoptoticproteins contains only the BH3 domain, implying that this particulardomain may be uniquely important in the promotion of apoptosis (Diaz etal., 1997).

[0075] In vivo Bax homodimerizes and also forms heterodimers with BCL-2,and overexpressed Bax overrides the death repressor activity of BCL-2(Oltvai et al., 1993). Bax expression levels higher than Bcl-2expression levels in bladder tumors correlates to an improved patientprognosis. In patients whose tumors expressed more Bcl-2 than Bax mRNA,early relapses were much more frequently observed (Gazzaniga et al.,1996).

[0076] Recently it was reported that a splice variant of Bax, Bax-alpha,was expressed in high amount in normal breast epithelium, whereas onlyweak or no expression was detected in 39 out of 40 cancer tissue samplesexamined (Bargou et al., 1996), and downregulation of Bax-alpha wasdetected in different histological subtypes. Furthermore, when Bax-alphawas transfected into breast cancer cell lines under the control of atetracycline-dependent expression system, Bax restored sensitivity ofthe cancer cells toward both serum starvation and APO-I/Fas-triggeredapoptosis, significantly reducing tumor growth in SCID mice. Therefore,it was proposed that disruption of apoptosis pathway may contribute tothe pathogenesis of breast cancer at least in part due to an imbalancebetween members of the Bcl-2 gene family (Bargou et al., 1996).

[0077] Additional members of the Bcl-2 family of apoptosis-inducingproteins have been identified. Bak, a new member of the Bcl-2 family, isexpressed in a wide variety of cell types and binds to the Bcl-2homologue Bcl-x2 in yeast (Farrow et al., 1995; Chittenden et al.,1995). A domain in Bak was identified as both necessary and sufficientfor cytotoxicity activity and binding to Bcl-x1. Furthermore, sequencessimilar to this domain that are distinct from BH1 and BH2 have beenidentified in Bax and Bipl. This domain is critical for mediating thefunction of multiple cell death-regulatory proteins that interact withBcl-2 family members (Chittenden et al., 1995).

[0078] Overexpression of Bak in sympathetic neurons deprived of nervegrowth factor accelerated apoptosis and blocked the protective effect ofco-injected E1B 19K. The adenovirus E1B 19K protein is known to inhibitapoptosis induced by E1A, tumor-necrosis factor-alpha, FAS antigen andnerve growth factor deprivation (Farrow et al., 1995). Expression of Bakinduced rapid and extensive apoptosis of serum-deprived fibroblasts,which suggests that Bak is directly involved in activating the celldeath machinery (Chittenden et al., 1995). In the normal and neoplasticcolon, mucosal expression of immunoreactive Bak co-localized with sitesof epithelial cell apoptosis. Induction of apoptosis in the human coloncancer cell line HT29 and the rat normal small intestinal cell line 1EC18 in culture was accompanied by increased Bak expression withoutconsistent changes in expression of other Bcl-2 homologous proteins(Moss et al., 1996). Therefore, Bak was also suggested to be theendogenous Bcl-2 family member best correlated with intestinal cellapoptosis (Moss et al., 1996).

[0079] Unlike Bax, however, Bak can inhibit cell death in anEpstein-Barr-virus-transformed cell line. Tissues with uniquedistribution of Bak messenger RNA include those containing long-lived,terminally differentiated cell types (Krajewski, et al., 1996),suggesting that cell-death-inducing activity is broadly distributed, andthat tissue-specific modulation of apoptosis is controlled primarily byregulation of molecules that inhibit apoptosis (Kiefer et al., 1995).

[0080] Another member of the Bcl2 family, Bad, possesses the key aminoacid motifs of BH1 and BH2 domains. Bad lacks the classical C-terminalsignal-anchor sequence responsible for the integral membrane positionsof other family members. Bad selectively dimerizes with Bcl-X_(L) aswell as Bcl-2, but not with Bax, Bcl-Xs-Mcl1, A1 or itself. Bad reversesthe death repressor activity of Bcl-X_(L), but not that of Bcl-2 (Yanget al., 1995; Ottilie et al., 1997; Zha et al., 1997).

[0081] Bik, another member of the Bcl-2 family, interacts with thecellular survival-promoting proteins, Bcl-2 and Bcl-X_(L) as well as theviral survival-promoting proteins, Epstein Barr virus-BHRF1 andadenovirus E1B-19kDa. In transient transfection assays, Bik promotescell death in a manner similar to Bax and Bak, other pro-apoptoticmembers of the Bcl-2 family. This pro-apoptosis activity of Bik can besuppressed by coexpression of Bcl-2, Bcl-X_(L), EBV-BHRF1 and E1B-19 kDaproteins, which suggests that Bik may be a common target for bothcellular and viral anti-apoptotic proteins. While Bik does not containovert homology to the BH1 and BH2 conserved domains characteristic ofthe Bcl-2 family, it shares a 9 amino acid domain (BH3) with Bax andBak, which may be a critical determinant for the death-promotingactivity of these proteins (Boyd et al., 1995; Han et al., 1996).

[0082] The Bcl-2 family is composed of various pairs of antagonist andagonist proteins that regulate apoptosis, although whether theirfunction is interdependent remains unclear. Utilizing gain-and loss offunction models of Bcl-2 and Bax, Knudson et al. (1997), demonstratedthat apoptosis and thymic hypoplasia, characteristic of Bcl-2-deficientmice, are largely absent in mice also deficient in Bax. A single copy ofBax promoted apoptosis in the absence of Bcl-2. However, overexpressionof Bcl-2 still repressed apoptosis in the absence of Bax. While an invivo competition exists between Bax and Bcl-2, each is able to regulateapoptosis independently. Bax has been shown to form channels in lipidmembranes and trigger the release of liposome-encapsulatedcarboxyluorescein at both neutral and acidic pH. At physiological pH,release could be blocked by Bcl-2. In planer lipid bilayers, Bax formedpH- and voltage-dependent ion-conduction channels. Thus, thepro-apoptotic effects of Bax may be elicited through an intrinsicpore-forming activity that can be antagonized by Bcl-2 (Antonsson etal., 1997). Two other members of this family, Bcl-2 and Bcl-1, were alsoshown to form pores in lipid membranes (Schendel et al., 1997).

[0083] II. Granzyme B and Apoptosis

[0084] Host defenses against viruses, parasitic agents, and transformedcells require cytotoxic T lymphocytes (CTLs) and natural killer (NK)cells (Berke, 1995; Kagi et al., 1996), which induce apoptosis in targetcells using at least two separate mechanisms. In the first mechanism,there is stimulation of cell surface death receptors (such as Fas) onthe target cells by death ligands expressed on the surface of theeffector cell (Nagata and Golstein, 1995; Ashkenazi and Dixit, 1998),which then leads to activation of caspase cascades in the target cell.In the second mechanism, denoted “granule exocytosis,” there is vectoraltransfer of the contents of effector cell cytoplasmic granules into thetarget cell (Doherty, 1993; Shresta et al., 1995a; Shresta et al.,1995b). Perforin and the granzyme family of serine proteases areimportant components of these granules.

[0085] Perforin is a 70 kDa protein that binds in a calcium-dependentmanner to membrane phosphorylcholine groups (Masson and Tshopp, 1985;Young et al., 1986; Tschopp et al., 1989). Subsequent to binding,perforin inserts into the membrane and oligomerizes, resulting in theformation of pores. This permeabilization of the membrane likely makespossible the entry of other molecules, such as granzymes, into thetarget cell.

[0086] Granzymes A and B are particularly abundant (Smyth et al., 1996)within the granules of CTLs and NK cells. Granzyme B, which is alsocalled fragmentin or cytotoxic T cell protease (CCP), is similar tocaspases having the characteristic of cleaving substrate proteins afteraspartate residues (Zunino et al., 1990; Lobe et al., 1986; Odake etal., 1991; Poe et al., 1991; Shi et al., 1992). Mice that are granzyme Bknockouts demonstrate an important role for granzyme B in the inductionof target cell apoptosis. CTLs and NK cells derived from granzymeB^(−/−) mice have a severely reduced capacity to induce apoptotic DNAfragmentation in target cells (Shresta et al., 1995a; Heusel et al.,1994). Although earlier complementary studies showed that purifiedgranzyme B alone did not promote apoptosis when added to target cells,cotreatment with purified granzyme B and perforin proteins inducedmarked DNA fragmentation and apoptotic features in four lymphoma targetcell lines (Shi et al., 1992). Therefore, it is possible that granzyme Bgains entry into target cells through perforin-generated pores, althoughthis is controversial. Several studies have shown that granzyme B isinternalized by target cells in the absence of added perforin (Froelichet al., 1996; Jans et al., 1996; Shi et al., 1997; Pinkoski et al.,1998; Pinkoski et al., 2000). The internalized granzyme B has beenreported to reside in the cytoplasm, (Jans et al., 1996; Shi et al.,1997) or in a novel vesicular compartment. (Pinkoski et al., 1998),although the triggering of apoptosis in cells that have internalizedgranzyme B requires further addition of perforin to the cells (Froelichet al., 1996; Jans et al., 1996; Shi et al., 1997; Pinkoski et al.,1998; Pinkoski et al., 2000). It is possible that perforin is requiredfor the release of granzyme B to the target cell from internal vesicles.Other studies have indicated that perforin facilitates translocation ofgranzyme B to the nucleus, and that nuclear localization is critical tothe ability of granzyme B to cause apoptosis (Jans et al., 1996; Shi etal., 1997; Pinkoski et al., 1998; Pinkoski et al., 2000; Pinkoski etal., 1996; Trapani et al., 1996).

[0087] Although the importance of granzyme B subcellular localizationremains controversial, it is certain that granzyme B has the ability toaffect the caspase pathway of apoptosis. In vitro studies have shownthat granzyme B is capable of cleaving procaspase3, -6, -7,-8m -9 and-10, (Darmon et al., 1996; Darmon et al., 1996; Martin et al., 1996;Quan et al., 1996; Fernandes-Alnemri et al., 1995; Orth et al., 1996;Fernandes-Alnemri et al., 1995; Chinnaiyan et al., 1996; Gu et al.,1996; Boldin et al., 1996; Muzio et al., 1996; Duan et al., 1996;Fernandes-Alnemri et al., 1996; Medema et al., 1997; Van de Craen etal., 1997; Talanian et al., 1997). In the case of procaspases-3, -7 and-9, granzyme B-mediated processing has been shown to generate activecaspase enzymes (Darmon et al., 1995; Quan et al., 1996; Gu et al.,1996; Duan et al., 1996). More importantly, studies with whole cellshave shown that caspases are activated in target cells followingcoincubation with granzyme B and perforin (Darmon et al., 1996; Talanianet al., 1997; Shi et al., 1996). It remains to be determined, however,which caspases are the preferred in vivo substrates for granzyme B. Inany event, it is reasonable to propose that granzyme B may promoteapoptosis simply by cleaving and activating endogenous caspases in thetarget cell.

[0088] Cleavage of the caspase substrate proteins PARP, lamin B, andU1-70 kDa is also observed in cells undergoing granzymeB/perforin-mediated apoptosis (Medema et al., 1997; Talanian et al.,1997; Shi et al., 1996; Andrade et al., 1998). These cleavage events arelikely due to caspases activated by cleavage by granzyme B, sincecleavage of all three proteins is inhibited by 100 μM DEVD- orVAD-containing peptides, which inhibit caspases, but not granzyme B(Darmon et al., 1996; Medema et al., 1997; Talanian et al,.1997; Shi etal., 1996; Andrade et al., 1998). Two additional caspase substrateproteins, DNA-PK_(CS) and NuMA, are also cleaved in granzymeB/perforin-treated cells, but cleavage of these proteins is insensitiveto DEVD or VAD peptide inhibitors (Andrade et al., 1998) Moreover, thesizes of the DNA-PK_(CS) and NuMA proteolytic fragments generated bygranzyme B differ from those resulting from caspase cleavage, whichsuggests that during granzyme B-mediated apoptosis, important cellularsubstrates are cleaved in a caspase-independent manner. The significanceof these caspase-independent cleavage events is unknown. However, giventhat granzyme B/perforin-mediated DNA fragmentation and apoptotic deathis significantly delayed by 100 μM DEVD/VAD, (Darmon et al., 1996;Talanian et al., 1997; Shi et al., 1996) this emphasizes the necessityfor caspase activation during this form of apoptosis.

[0089] III. Granzyme A and Apoptosis

[0090] The mature granzyme A enzyme is a disulphide cross-linkedhomodimer of 50 kDa that cleaves substrate proteins following lysine orarginine residues (Odake et al., 1991; Gershenfeld et al., 1986; Massonet al., 1986), and granzyme A is the most abundant protease found in thegranules of CTL cells. The mechanism of action of this protease differssignificantly from that of granzyme B, although granzyme A is capable ofinducing apoptosis after loading into target cells. Furthermore, it isthought that the role of granzyme A in CTL induced apoptosis issignificantly more subtle than that of granzyme B. For example, micewhich are deficient in granzyme A expression (granzyme A^(−/−) mice)exhibit relatively normal CTL-mediated cytotoxicity (Andrade et al.,1998), although they are unable to clear the mouse pox virus Ectromelia(Mulbacher et al., 1996). In contrast, CTLs from granzyme B^(−/−) miceare capable of inducing target cell death only after prolongedcoincubation (Heusel et al., 1994), and, therefore, granzyme B iscritically important for rapid CTL killing. Recent experiments usingmice deficient in both granzyme A and granzyme B suggest that granzyme Adoes have some role in CTL-mediated killing. CTLs from granzyme A^(−/−)/granzyme B^(−/−) mice are unable to induce target cell DNAfragmentation, even after prolonged coincubation (Shresta et al., 1999),which indicates that granzyme A activity accounts for the ability ofgranzyme B^(−/−) CTLs to induce target cell apoptosis followingprolonged exposure. Therefore, granzyme A may allow CTLs to kill targetcells under conditions where granzyme B activity is inhibited (e.g.target cells that express granzyme B inhibitors).

[0091] In studies with recombinant proteins, coincubation of granzyme Aand perforin with target cells leads to rapid (within 2 hours)accumulation of DNA single-strand breaks (Hayes et al., 1980; Beresfordet al., 1999), which contrasts with the rapid degradation of DNA tooligonucleosomal-length fragments seen in cells treated with granzyme Band perforin. Granzyme A/perforin treatment also leads to nuclearcondensation (Beresford et al., 1999). These effects which occur inresponse to granzyme A are insensitive to caspase inhibitors, indicatingthat these actions of granzyme A are caspase-independent (Beresford etal., 1999). In a consistent manner, granzyme A/perforin treatment doesnot result in processing/activation of procaspase-3 or cleavage of thecaspase substrate proteins PARP, laminB, or rho-GTPase (Beresford etal., 1999) However, granzymeB-induced DNA fragmentation is strictlydependent on the activation of caspases. Both granzyme A and granzyme B(in conjunction with perforin) also induce target cell cytolysis, bothcases of which are caspase-independent events. Thus, current evidenceindicates that granzyme B is the primary CTL mediator of target cell DNAfragmentation and apoptotic death, and that the apoptotic effects ofthis protease are mediated primarily through the activation of caspase.Alternatively, granzyme A may be more of a default or specializedmediator of target cell apoptosis, with the pathways initiated bygranzyme A being distinctly different from those initiated by granzymeB.

[0092] IV. Generation of Chimeric Molecules

[0093] While the chimeric proteins of the present invention may beproduced by chemical synthetic methods or by chemical linkage betweenthe two moieties, it is preferred that they are produced by fusion of acoding sequence of a cell-specific targeting moiety and a codingsequence of an apoptosis-inducing protein under the control of aregulatory sequence which directs the expression of the fusionpolynucleotide in an appropriate host cell. In preferred embodiments,each of the components of the chimeric protein comprise functionalactivity for their respective parts being a cell-specific targetingmoiety and a signal transduction pathway factor (such as anapoptosis-inducing protein).

[0094] The fusion of two full-length coding sequences can be achieved bymethods well known in the art of molecular biology. It is preferred thata fusion polynucleotide contain only the AUG translation initiationcodon at the 5′ end of the first coding sequence without the initiationcodon of the second coding sequence to avoid the production of twoseparate encoded products. In addition, a leader sequence may be placedat the 5′ end of the polynucleotide in order to target the expressedproduct to a specific site or compartment within a host cell tofacilitate secretion or subsequent purification after gene expression.The two coding sequences can be fused directly without any linker or byusing a flexible polylinker, such as one composed of the pentamerGly-Gly-Gly-Gly-Ser (SEQ ID NO:50) repeated 1 to 3 times. Such linkerhas been used in constructing single chain antibodies (scfv) by beinginserted between V_(H) and V_(L) (Bird et al., 1988; Huston et al.,1988). The linker is designed to enable the correct interaction betweentwo beta-sheets forming the variable region of the single chainantibody. Other linkers which may be used includeGlu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:51)(Chaudhary et al., 1990) andLys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg -Ser-Leu-Asp(SEQ ID NO:52)(Bird et al., 1988).

[0095] A. Cell-specific Targeting Moieties

[0096] The chimeric proteins of the invention are composed of acell-specific targeting moiety and an apoptosis-inducing moiety. Thecell-specific targeting moiety confers cell-type specific binding to themolecule, and it is chosen on the basis of the particular cellpopulation to be targeted. A wide variety of proteins are suitable foruse as cell-specific targeting moieties, including but not limited to,ligands for receptors such as growth factors, hormones and cytokines,and antibodies or antigen-binding fragments thereof.

[0097] Since a large number of cell surface receptors have beenidentified in hematopoietic cells of various lineages, ligands orantibodies specific for these receptors may be used as cell-specifictargeting moieties. IL2 may be used as a cell-specific targeting moietyin a chimeric protein to target IL2R⁺cells. Alternatively, othermolecules such as B7-1, B7-2 and CD40 may be used to specifically targetactivated T cells (The Leucocyte Antigen Facts Book, 1993, Barclay etal. (eds.), Academic Press). Furthermore, B cells express CD19, CD40 andIL4 receptor and may be targeted by moieties that bind these receptors,such as CD40 ligand, IL4, IL5, IL6 and CD28. The elimination of immunecells such as T cells and B cells is particularly useful in thetreatment of autoimmunity, hypersensitivity, transplantation rejectionresponses and in the treatment of lymphoid tumors. Examples ofautoimmune diseases are multiple sclerosis, rheumatoid arthritis,insulin-dependent diabetes mellitus, systemic lupus crythemotisis,scleroderma, and uviatis. More specifically, since myelin basic proteinis known to be the major target of immune cell attack in multiplesclerosis, this protein may be used as a cell-specific targeting moietyfor the treatment of multiple sclerosis (WO 97/19179; Becker et al.,1997).

[0098] Other cytokines that may be used to target specific cell subsetsinclude the interleukins (IL1 through IL15), granulocyte-colonystimulating factor, macrophage-colony stimulating factor,granulocyte-macrophage colony stimulating factor, leukemia inhibitoryfactor, tumor necrosis factor, transforming growth factor, epidermalgrowth factor, insulin-like growth factors, and/or fibroblast growthfactor (Thompson (ed.), 1994, The Cytokine Handbook, Academic Press, SanDiego).

[0099] A skilled artisan recognizes that there are a variety of knowncytokines, including hematopoietins (four-helix bundles) (such as Epo(erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF),IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6 IL-4 (IFN-β₂, BSF-2, BCDF),IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T-cell growfactor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM(OM, oncostatin M), and LIF (leukemia inhibitory factor)); interferons(such as IFN-γ, IFN-α, and IFN-β); immunoglobin superfamily (such asB7.1 (CD80), and B7.2 (B70, CD86)); TNF family (such as TNF-α(cachectin), TNF-β (lymphotoxin, LT, LT-α), LT-β, CD40 ligand (CD40L),Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and4-1BBL)); and those unassigned to a particular family (such as TGF-β,IL-1α, IL-1β, IL-1 RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NKcell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and/or IL-18(IGIF, interferon-γ inducing factor)).

[0100] Additionally, certain cell surface molecules are highly expressedin tumor cells, including hormone receptors such as human chorionicgonadotropin receptor and gonadotropin releasing hormone receptor(Nechushtan et al., 1997). Therefore, the corresponding hormones may beused as the cell-specific targeting moieties in cancer therapy.

[0101] Thus, in some embodiments of the invention, no antibodies areutilized in the chimeric polypeptides. However, antibodies are extremelyversatile and useful cell-specific targeting moieties because they canbe generated against any cell surface antigen of interest. Monoclonalantibodies have been generated against cell surface receptors,tumor-associated antigens, and leukocyte lineage-specific markers suchas CD antigens. Antibody variable region genes can be readily isolatedfrom hybridoma cells by methods well known in the art.

[0102] Over the past few years, several monoclonal antibodies have beenapproved for therapeutic use and have achieved significant clinical andcommercial success. Much of the clinical utility of monoclonalantibodies results from the affinity and specificity with which theybind to their targets, as well as long circulating life due to theirrelatively large size. Monoclonal antibodies, however, are not wellsuited for use in indications where a short half-life is advantageous orwhere their large size inhibits them physically from reaching the areaof potential therapeutic activity.

[0103] Moreover, antibodies in their native form, consisting of twodifferent polypeptide chains that need to be generated in approximatelyequal amounts and assembled correctly, are poor candidates fortherapeutic purposes. However, it is possible to create a singlepolypeptide which can retain the antigen binding properties of amonoclonal antibody.

[0104] Single chain antibodies (SCAs) are genetically engineeredproteins designed to expand on the therapeutic and diagnosticapplications possible with monoclonal antibodies. SCAs have the bindingspecificity and affinity of monoclonal antibodies and, in their nativeform, are about one-fifth to one-sixth of the size of a monoclonalantibody, typically giving them very short half-lives. Human SCAs offermany benefits compared to most monoclonal antibodies, including morespecific localization to target sites in the body, faster clearance fromthe body, and a better opportunity to be used orally, intranasally,transdermally or by inhalation. In addition to these benefits,fully-human SCAs can be isolated directly from human SCA librarieswithout the need for costly and time consuming “humanization”procedures. SCAs are also readily produced through intracellularexpression (inside cells) allowing for their use in gene therapyapplications where SCA molecules act as specific inhibitors of cellfunction.

[0105] The variable regions from the heavy and light chains (VH and VL)are both approximately 110 amino acids long. They can be linked by a 15amino acid linker with the sequence (SEQ ID NO:50)₃, which hassufficient flexibility to allow the two domains to assemble a functionalantigen binding pocket. In specific embodiments, addition of varioussignal sequences allows the scFv to be targeted to different organelleswithin the cell, or to be secreted. Addition of the light chain constantregion (Ck) allows dimerization via disulfide bonds, giving increasedstability and avidity. Thus, for a single chain Fv (scFv) SCA, althoughthe two domains of the Fv fragment are coded for by separate genes, ithas been proven possible to make a synthetic linker that enables them tobe made as a single protein chain scFv (Bird et al., 1988; Huston etal., 1988) by recombinant methods. Furthermore, they are frequently useddue to their ease of isolation from phage display libraries and theirability to recognize conserved antigens (for review, see Adams andSchier, 1999). For example, scFv is utilized to target suicide genes tocarcinoembryonic antigen (CEA)-expressing tumor cells by a retrovectordisplaying anti-CEA scFv (Kuroki et al., 2000).

[0106] Finally, the Fc portion of the heavy chain of an antibody may beused to target Fc receptor-expressing cells such as the use of the Fcportion of an IgE antibody to target mast cells and basophils. The useof antibodies to target a polypeptide or peptide of interest byantibody-directed therapy or immunological-directed therapy is currentlyapproved and in use in the present therapeutic market.

[0107] Thus, it is preferred that a scFv be used as a cell-specifictargeting moiety in the present invention.

[0108] B. Apoptosis-inducing Moieties

[0109] The pro-apoptotic proteins in the BCL2 family are particularlysuitable for use as the apoptosis-inducing moieties in the presentinvention. Such human proteins are expected to have reducedimmunogenicity over many immunotoxins composed of bacterial toxins.Although Bax is a useful apoptosis-inducing moiety in one embodiment ofthe present invention, other members in this family are suitable for usein the present invention and include Bak (Farrow et al., 1995;Chittenden et al., 1995; Kiefer et al., 1995), Bcl-X_(s), (Boise et al.,1993; Fang et al., 1994), Bad (Yang et al., 1995), Bid (Wang et al.,1996), Bik (Boyd et al., 1995), Hrk (Inohara et al., 1997) and/or Bok(Hsu et al., 1997). The nucleotide sequences encoding these proteins areknown in the art and are readily obtainable from databases such asGenBank, and thus CDNA clones can be readily obtained for fusion with acoding sequence for a cell-specific targeting moiety in an expressionvector.

[0110] Specific domains of particular members of the Bcl-2 family havebeen studied regarding their apoptosis-inducing activities. For example,the GD domain of Bak is involved in the apoptosis function (U.S. Pat.No. 5,656,725). In addition, Bax and Bip1a share a homologous domain.Therefore, any biologically active domains of the Bcl-2 family may beused as an apoptosis-inducing moiety for the practice of the presentinvention.

[0111] Caspases also play a central role in apoptosis and may wellconstitute part of the consensus core mechanism of apoptosis. Caspasesare implicated as mediators of apoptosis. Since the recognition thatCED-3, a protein required for developmental cell death, has sequenceidentity with the mammalian cysteine protease interleukin-1beta-converting enzyme (ICE), a family of at least 10 related cysteineproteases has been identified. These proteins are characterized byalmost absolute specificity for aspartic acid in the PI position. Allthe caspases (ICE-like proteases) contain a conserved QACKG (where X isR, Z or G) pentapeptide active-site motif. Caspases are synthesized asinactive proenzymes comprising an N-terminal peptide (Prodomain)together with one large and one small subunit. The crystal structures ofboth caspase-1 and caspase-3 show that the active enzyme is aheterotetramer, containing two small and two large subunits. Activationof caspases during apoptosis results in the cleavage of criticalcellular substrates, including poly (ADP-riose) polymerase and lamins,so precipitating the dramatic morphological changes of apoptosis (Cohen,1997, Biochem. J. 326:1-16). Therefore, it is also within the scope ofthe present invention to use a caspase as an apoptosis-inducing moiety.

[0112] Recently a few new proteins were cloned and identified as factorsrequired for mediating activity of proteins, mainly caspases, involvedin the apoptosis pathway. One factor was identified as the previouslyknown electron transfer protein, cytochrome c (Lin et al., 1996, Cell86:147-157), designed as Apaf-2. In addition to cytochrome c theactivation of caspase-3 requires two other cytosolic factors-Apaf-1 andApaf-3. Apaf-1 is a protein homologous to C. elegans CED-4, and Apaf-3was identified as a member of the caspase family, caspase-9. Bothfactors bind to each other via their respective NH2-terminal CED-3homologous domains, in the presence of cytochrome c, an event that leadsto caspase-9 activation. Activated caspase-9 in turn cleaves andactivates caspase-3 (Liu et al., 1996; Zou et al., 1997; Li et al.,1997). Another protein involved in the apoptotic pathway is DNAfragmentation factor (DFF), a heterodimer of 45 and 40 kd subunits thatfunctions downstream of caspase-3 to trigger fragmentation of genomicDNA into nucleosomal segments (Liu et al., 1997).

[0113] C. Chimeric Polypeptide Production

[0114] In accordance with the objects of the present invention, apolynucleotide that encodes a chimeric protein, mutant polypeptide,biologically active fragment of chimeric protein, or functionalequivalent thereof, may be used to generate recombinant DNA moleculesthat direct the expression of the chimeric protein, chimeric peptidefragments, or a functional equivalent thereof, in appropriate hostcells.

[0115] Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence, may be used in the practice of theinvention of the cloning and expression of the chimeric protein. SuchDNA sequences include those capable of hybridizing to the chimericsequences or their complementary sequences under stringent conditions.In one embodiment, the phrase “stringent conditions” as used hereinrefers to those hybridizing conditions that (1) employ low ionicstrength and high temperature for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with a 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

[0116] Altered DNA sequences that may be used in accordance with theinvention include deletions, additions or substitutions of differentnucleotide residues resulting in a sequence that encodes the same or afunctionally equivalent fusion gene product. The gene product itself maycontain deletions, additions or substitutions of amino acid residueswithin a chimeric sequence, which result in a silent change thusproducing a functionally equivalent chimeric protein. Such amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine, histidine and arginine; amino acidswith uncharged polar head groups having similar hydrophilicity valuesinclude the following: glycine, asparagine, glutamine, serine,threonine, tyrosine; and amino acids with nonpolar head groups includealanine, valine, isoleucine, leucine, phenylalanine, proline,methionine, tryptophan.

[0117] The DNA sequences of the invention may be engineered in order toalter a chimeric coding sequence for a variety of ends, including butnot limited to, alterations which modify processing and expression ofthe gene product. For example, mutations may be introduced usingtechniques which are well known in the art, e.g., site-directedmutagenesis, to insert new restriction sites, to alter glycosylationpatterns, phosphorylation, etc.

[0118] In an alternate embodiment of the invention, the coding sequenceof the chimeric protein could be synthesized in whole or in part, usingchemical methods well known in the art. (See, for example, Caruthers etal., 1980; Crea and Horn, 1980; and Chow and Kempe, 1981). For example,active domains of the moieties can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography followed by chemical linkage to form achimeric protein. (e.g., see Creighton, 1983, Proteins Structures AndMolecular Principles, W. H. Freeman and Co., N.Y. pp. 50-60). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W. H.Freeman and Co., N.Y. pp. 34-49). Alternatively, the two moieties of thechimeric protein produced by synthetic or recombinant methods may beconjugated by chemical linkers according to methods well known in theart (Brinkmann and Pastan, 1994).

[0119] In order to express a biologically active chimeric protein, thenucleotide sequence coding for a chimeric protein, or a functionalequivalent, is inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. The chimeric gene productsas well as host cells or cell lines transfected or transformed withrecombinant chimeric expression vectors can be used for a variety ofpurposes. These include but are not limited to generating antibodies(i.e., monoclonal or polyclonal) that bind to epitopes of the proteinsto facilitate their purification.

[0120] Methods that are well known to those skilled in the art can beused to construct expression vectors containing the chimeric proteincoding sequence and appropriate transcriptional/translational controlsignals. These methods include in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Sambrook et al., 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.

[0121] A variety of host-expression vector systems may be utilized toexpress the chimeric protein coding sequence. These include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the chimeric protein coding sequence; yeast transformed withrecombinant yeast expression vectors containing the chimeric proteincoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the chimeric proteincoding sequence; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing the chimeric protein coding sequence; oranimal cell systems. It should be noted that since mostapoptosis-inducing proteins cause programmed cell death in mammaliancells, it is preferred that the chimeric protein of the invention beexpressed in prokaryotic or lower eukaryotic cells. Section 6illustrates that IL2-Bax may be efficiently expressed in E. coli.

[0122] The expression elements of each system vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter; cytomegalovirus promoter) and the like may be used;when cloning in insect cell systems, promoters such as the baculoviruspolyhedrin promoter may be used; when cloning in plant cell systems,promoters derived from the genome of plant cells (e.g., heat shockpromoters; the promoter for the small subunit of RUBISCO; the promoterfor the chlorophyll α/β binding protein) or from plant viruses (e.g.,the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may beused; when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter) may be used; when generating cell lines thatcontain multiple copies of the chimeric DNA, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

[0123] In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the chimericprotein expressed. For example, when large quantities of chimericprotein are to be produced, vectors which direct the expression of highlevels of protein products that are readily purified may be desirable.Such vectors include but are not limited to the pHL906 vector (Fishmanet al., 1994), the E. coli expression vector pUR278 (Ruther et al.,1983), in which the chimeric protein coding sequence may be ligated intothe vector in frame with the lacZ coding region so that a hybrid AS-lacZprotein is produced; pIN vectors (Inouye and Inouye, 1989; Van Heeke andSchuster, 1989); and the like.

[0124] An alternative expression system which could be used to expresschimeric protein is an insect system. In one such system, Autographacalifornica nuclear polyhidrosis virus (AcNPV) is used as a vector toexpress foreign genes. The virus grows in Spodoptera frugiperda cells.The chimeric protein coding sequence may be cloned into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of the chimeric protein coding sequence will resultin inactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted gene isexpressed. (e.g., see Smith et al., 1983; U.S. Pat. No. 4,215,051).

[0125] Specific initiation signals may also be required for efficienttranslation of the inserted chimeric protein coding sequence. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where the entire chimeric gene, including its own initiation codonand adjacent sequences, is inserted into the appropriate expressionvector, no additional translational control signals may be needed.However, in cases where the chimeric protein coding sequence does notinclude its own initiation codon, exogenous translational controlsignals, including the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the chimeric protein coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987).

[0126] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. The presence of consensusN-glycosylation sites in a chimeric protein may require propermodification for optimal chimeric protein function. Different host cellshave characteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the chimeric protein. To this end, eukaryotic host cells whichpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the chimeric proteinmay be used. Such mammalian host cells include but are not limited toCHO, VERO, BHK, HeLa, COS, MDCK, 293, W138, and the like.

[0127] For long-term, high-yield production of recombinant chimericproteins, stable expression is preferred. For example, cell lines whichstably express the chimeric protein may be engineered. Rather than usingexpression vectors which contain viral originals of replication, hostcells can be transformed with a chimeric coding sequence controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines.

[0128] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalski andSzybalski, 1962), and adenine phosphoribosyltransferase (Lowy et al.,1980) genes can be employed in tk-, hgprt- or aprt-cells, respectively.Also, antimetabolite resistance can be used as the basis of selectionfor dhfr, which confers resistance to methotrexate (Wigler et al., 1980;O'Hare et al., 1981); gpt, which confers resistance to mycophenolic acid(Mulligan and Berg, 1981); neo, which confers resistance to theaminoglycoside G-418 (Colbere-Garapin et al., 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984) genes.Additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman andMulligan, 1988); and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

[0129] D. Protein Purification

[0130] The chimeric proteins of the invention can be purified byart-known techniques such as high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, affinity chromatographyand the like. The actual conditions used to purify a particular proteinwill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity, etc., and will be apparent to those having skill in theart.

[0131] For affinity chromatography purification, any antibody whichspecifically binds the protein may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a chimeric proteinor a fragment thereof. The protein may be attached to a suitablecarrier, such as bovine serum albumin (BSA), by means of a side chainfunctional group or linkers attached to a side chain functional group.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhold limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmetter-Guerin) and Corynebacterium parvum.

[0132] Monoclonal antibodies to a chimeric protein may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(1975), the human B-cell hybridoma technique, (Kosbor et al., 1983; Coteet al., 1983) and the EBV-hybridoma technique (Cole et al., 1985). Inaddition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984; Neuberger et al., 1984; Takeda etal., 1985) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used.Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce chimericprotein-specific single chain antibodies for chimeric proteinpurification and detection.

[0133] V. Uses of Chimeric Polypeptide

[0134] Once a chimeric protein is expressed and purified, its identityand functional activities can be readily determined by methods wellknown in the art. For example, antibodies to the two moieties of theprotein may be used to identify the protein in Western blot analysis. Inaddition, the chimeric protein can be tested for specific binding totarget cells in binding assays using a fluorescent-labeled orradiolabelled secondary antibody.

[0135] A. In vitro and Ex vivo Uses

[0136] The chimeric polypeptides of the invention are useful fortargeting specific cell types in a cell mixture, and eliminating thetarget cells by inducing apoptosis. The chimeric polypeptides of theinvention are also useful as a diagnostic reagent. The binding of achimeric protein to a target cell can be readily detected by using asecondary antibody specific for the apoptosis-inducing moiety. In thatconnection, the secondary antibody or the chimeric protein enzyme or aradioisotope to facilitate the detection of binding of the chimericprotein to a cell.

[0137] B. In vivo Uses

[0138] In some embodiments, an effective amount of the chimericpolypeptides of the present invention are administered to a cell. Inother embodiments, a therapeutically effective amount of the chimericpolypeptides of the present invention are administered to an individualfor the treatment of disease. The term “effective amount” as used hereinis defined as the amount of the chimeric polypeptides of the presentinvention which are necessary to result in a physiological change in thecell or tissue to which it is administered. The term “therapeuticallyeffective amount” as used herein is defined as the amount of thechimeric polypeptides of the present invention that eliminate, decrease,delay, or minimize adverse effects of a disease, such as cancer. Askilled artisan readily recognizes that in many cases the chimericpolypeptide may not provide a cure but may only provide partial benefit.In some embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of chimeric polypeptide that provides a physiological change isconsidered an “effective amount” or a “therapeutically effectiveamount.”

[0139] The chimeric proteins of the invention may be administered to asubject per se or in the form of a pharmaceutical composition for thetreatment of cancer, autoimmunity, transplantation rejection,post-traumatic immune responses and infectious diseases by targetingviral antigens, such as gp120 of HIV. More specifically, the chimericpolypeptides may be useful in eliminating cells involved in immunecell-mediated disorder, including lymphoma; autoimmunity,transplantation rejection, graft-versus-host disease, ischemia andstroke. Pharmaceutical compositions comprising the proteins of theinvention may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the proteins into preparations which canbe used pharmaceutically. Proper formulation is dependent upon the routeof administration chosen.

[0140] For topical administration the proteins of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

[0141] Systemic formulations include those designed for administrationby injection, e.g. subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, inhalation, oral or pulmonary administration.

[0142] For injection, the proteins of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0143] Alternatively, the proteins may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0144] For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0145] For oral administration, the proteins can be readily formulatedby combining the proteins with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the proteins of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. For oral solid formulations such as, for example,powders, capsules and tablets, suitable excipients include fillers suchas sugars, e.g. lactose, sucrose, mannitol and sorbitol; cellulosepreparations such as maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

[0146] If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques.

[0147] For oral liquid preparations such as, for example, suspensions,elixirs and solutions, suitable carriers, excipients or diluents includewater, glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

[0148] For buccal administration, the proteins may take the form oftablets, lozenges, etc. formulated in conventional manner.

[0149] For administration by inhalation, the proteins for use accordingto the present invention are conveniently delivered in the form of anaerosol spray from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of gelatin for use in an inhaler or insufflatormay be formulated containing a powder mix of the protein and a suitablepowder base such as lactose or starch.

[0150] The proteins may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

[0151] In addition to the formulations described previously, theproteins may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the proteins may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0152] Alternatively, other pharmaceutical delivery systems may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles that may be used to deliver proteins of the invention. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, theproteins may be delivered using a sustained-release system, such assemipermeable matrices of solid polymers containing the therapeuticagent. Various of sustained-release materials have been established andare well known by those skilled in the art. Sustained-release capsulesmay, depending on their chemical nature, release the proteins for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the chimeric protein, additional strategies forprotein stabilization may be employed.

[0153] As the proteins of the invention may contain charged side chainsor termini, they may be included in any of the above-describedformulations as the free acids or bases or as pharmaceuticallyacceptable salts. Pharmaceutically acceptable salts are those saltswhich substantially retain the biologic activity of the free bases andwhich are prepared by reaction with inorganic acids. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

[0154] 1. Effective Dosages

[0155] The proteins of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the proteins of the invention, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. A therapeutically effective amount is an amounteffective to ameliorate or prevent the symptoms, or prolong the survivalof, the patient being treated. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

[0156] For systemic administration, a therapeutically effective dose canbe estimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

[0157] Initial dosages can also be estimated from in vivo data, e.g.,animal models, using techniques that are well known in the art. Onehaving ordinary skill in the art could readily optimize administrationto humans based on animal data.

[0158] Dosage amount and interval may be adjusted individually toprovide plasma levels of the proteins which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

[0159] In cases of local administration or selective uptake, theeffective local concentration of the proteins may not be related toplasma concentration. One having skill in the art will be able tooptimize therapeutically effective local dosages without undueexperimentation.

[0160] The amount of protein administered will, of course, be dependenton the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration and the judgment of theprescribing physician.

[0161] The therapy may be repeated intermittently while symptomsdetectable or even when they are not detectable. The therapy may beprovided alone or in combination with other drugs. In the case ofautoimmune disorders, the drugs that may be used in combination withIL2-Bax of the invention include, but are not limited to, steroid andnon-steroid anti-inflammatory agents.

[0162] 2. Toxicity

[0163] Preferably, a therapeutically effective dose of the chimericproteins described herein will provide therapeutic benefit withoutcausing substantial toxicity.

[0164] Toxicity of the proteins described herein can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. Proteins which exhibit high therapeutic indices are preferred.The data obtained from these cell culture assays and animal studies canbe used in formulating a dosage range that is not toxic for use inhuman. The dosage of the proteins described herein lies preferablywithin a range of circulating concentrations that include the effectivedose with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basisof Therapeutics, Ch. 1, p. 1).

[0165] VI. Biological Functional Equivalents

[0166] As modifications and/or changes may be made in the structure ofthe polynucleotides encoding the chimeric polypeptides of the presentinvention and/or the chimeric polypeptides themselves according to thepresent invention, while obtaining molecules having similar or improvedcharacteristics, such biologically functional equivalents are alsoencompassed within the present invention.

[0167] A. Modified Polynucleotides and Polypeptides

[0168] The biological functional equivalent may comprise apolynucleotide that has been engineered to contain distinct sequenceswhile at the same time retaining the capacity to encode the “wild-type”or standard protein. This can be accomplished to the degeneracy of thegenetic code, i.e., the presence of multiple codons, which encode forthe same amino acids. In one example, one of skill in the art may wishto introduce a restriction enzyme recognition sequence into apolynucleotide while not disturbing the ability of that polynucleotideto encode a protein.

[0169] In another example, a polynucleotide encoding the chimericpolypeptide may be (and may encode) a biological functional equivalentwith more significant changes. Certain amino acids may be substitutedfor other amino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies, binding sites on substratemolecules, receptors, and the like. So-called “conservative” changes donot disrupt the biological activity of the polypeptide, as thestructural change is not one that impinges of the polypeptide's abilityto carry out its designed function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

[0170] In terms of functional equivalents, it is well understood by theskilled artisan that, inherent in the definition of a “biologicallyfunctional equivalent” protein and/or polynucleotide, is the conceptthat there is a limit to the number of changes that may be made within adefined portion of the molecule while retaining a molecule with anacceptable level of equivalent biological activity. Biologicallyfunctional equivalents are thus defined herein as those polypeptides(and polynucleotides) in selected amino acids (or codons) may besubstituted. Functional activity comprises the ability to kill a targetcell for the signal transduction pathway factor moiety or the ability totarget a cell specifically for the cell-specific targeting moiety.

[0171] In general, the shorter the length of the molecule, the fewerchanges that can be made within the molecule while retaining function.Longer domains may have an intermediate number of changes. Thefull-length protein will have the most tolerance for a larger number ofchanges. However, it must be appreciated that certain molecules ordomains that are highly dependent upon their structure may toleratelittle or no modification.

[0172] Amino acid substitutions are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are alla similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

[0173] To effect more quantitative changes, the hydropathic index ofamino acids may be considered. Each amino acid has been assigned ahydropathic index on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and/or arginine (−4.5).

[0174] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

[0175] It also is understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein and/orpeptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and/or antigenicity, i.e., with a biological property ofthe protein.

[0176] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0):threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4). In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, and/orthose within ±10.5 are even more particularly preferred.

[0177] B. Altered Amino Acids

[0178] The present invention, in many aspects, relies on the synthesisof peptides and polypeptides in cyto, via transcription and translationof appropriate polynucleotides. These peptides and polypeptides willinclude the twenty “natural” amino acids, and post-translationalmodifications thereof. However, in vitro peptide synthesis permits theuse of modified and/or unusual amino acids. A table of exemplary, butnot limiting, modified and/or unusual amino acids is provided hereinbelow. TABLE 2 Modified and/or Unusual Amino Acids Abbr. Amino AcidAbbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad3-Aminoadipic acid Hyl Hydroxylysine BAla beta-alanine,beta-Amino-propionic AHyl allo-Hydroxylysine acid Abu 2-Aminobutyricacid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid, piperidinic 4Hyp4-Hydroxyproline acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe2-Aminoheptanoic acid Aile allo-Isoleucine Aib 2-Aminoisobutyric acidMeGly N-Methylglycine, sarcosine BAib 3-Aminoisobutyric acid MeIleN-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva NorvalineDpm 2,2′-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionicacid Orn Ornithine EtGly N-Ethylglycine

[0179] C. Mimetics

[0180] In addition to the biological functional equivalents discussedabove, the present inventors also contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

[0181] Certain mimetics that mimic elements of protein secondary andtertiary structure are described in Johnson et al. (1993). Theunderlying rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains in such a way as to facilitate molecular interactions, such asthose of antibody and/or antigen. A peptide mimetic is thus designed topermit molecular interactions similar to the natural molecule.

[0182] Some successful applications of the peptide mimetic concept havefocused on mimetics of β-turns within proteins, which are known to behighly antigenic. Likely β-turn structure within a polypeptide can bepredicted by computer-based algorithms, as discussed herein. Once thecomponent amino acids of the turn are determined, mimetics can beconstructed to achieve a similar spatial orientation of the essentialelements of the amino acid side chains.

[0183] Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins. Vita et al. (1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

[0184] Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

[0185] Methods for generating specific structures have been disclosed inthe art. For example, alpha-helix mimetics are disclosed in U.S. Pat.Nos. 5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structuresrender the peptide or protein more thermally stable, also increaseresistance to proteolytic degradation. Six, seven, eleven, twelve,thirteen and fourteen membered ring structures are disclosed.

[0186] Methods for generating conformationally restricted beta turns andbeta bulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

[0187] D. Liposome Targeting

[0188] Association of the chimeric polypeptide with a liposome mayimprove biodistribution and other properties of the chimericpolypeptide. For example, liposome-mediated nucleic acid delivery andexpression of foreign DNA in vitro has been very successful (Nicolau andSene, 1982; Fraleyet al., 1979; Nicolauet al., 1987). The feasibility ofliposome-mediated delivery and expression of foreign DNA in culturedchick embryo, HeLa and hepatoma cells has also been demonstrated (Wonget al., 1980). Successful liposome-mediated gene transfer in rats afterintravenous injection has also been accomplished (Nicolau et al., 1987).

[0189] It is contemplated that a liposome/chimeric polypeptidecomposition may comprise additional materials for delivery to a tissue.For example, in certain embodiments of the invention, the lipid orliposome may be associated with a hemagglutinating virus (HVJ). This hasbeen shown to facilitate fusion with the cell membrane and promote cellentry of liposome-encapsulated DNA (Kaneda et al., 1989). In anotherexample, the lipid or liposome may be complexed or employed inconjunction with nuclear non-histone chromosomal proteins (HMG-1) (Katoet al., 1991). In yet further embodiments, the lipid may be complexed oremployed in conjunction with both HVJ and HMG-1.

[0190] Targeted delivery is achieved by the addition of ligands withoutcompromising the ability of these liposomes deliver large amounts ofchimeric polypeptide. It is contemplated that this will enable deliveryto specific cells, tissues and organs. The targeting specificity of theligand-based delivery systems are based on the distribution of theligand receptors on different cell types. The targeting ligand mayeither be non-covalently or covalently associated with the lipidcomplex, and can be conjugated to the liposomes by a variety of methods.

[0191] E. Cross-linkers

[0192] Bifunctional cross-linking reagents have been extensively usedfor a variety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies.Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

[0193] Exemplary methods for cross-linking ligands to liposomes aredescribed in U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511, eachspecifically incorporated herein by reference in its entirety). Variousligands can be covalently bound to liposomal surfaces through thecross-linking of amine residues. Liposomes, in particular, multilamellarvesicles (MLV) or unilamellar vesicles such as microemulsified liposomes(MEL) and large unilamellar liposomes (LUVET), each containingphosphatidylethanolamine (PE), have been prepared by establishedprocedures. The inclusion of PE in the liposome provides an activefunctional residue, a primary amine, on the liposomal surface forcross-linking purposes. Ligands such as epidermal growth factor (EGF)have been successfully linked with PE-liposomes. Ligands are boundcovalently to discrete sites on the liposome surfaces. The number andsurface density of these sites will be dictated by the liposomeformulation and the liposome type. The liposomal surfaces may also havesites for non-covalent association. To form covalent conjugates ofligands and liposomes, cross-linking reagents have been studied foreffectiveness and biocompatibility. Cross-linking reagents includeglutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycoldiglycidyl ether (EGDE), and a water soluble carbodiimide, preferably1-ethyl-3-(3-dimethylaminopropyl) carboddiimide (EDC). Through thecomplex chemistry of cross-linking, linkage of the amine residues of therecognizing substance and liposomes is established.

[0194] In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155, specifically incorporated herein by reference in itsentirety). The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides and sugars. Table 3 details certainhetero-bifunctional cross-linkers considered useful in the presentinvention. TABLE 3 HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm ReactiveLength\after Linker Toward Advantages and Applications cross-linkingSMPT Primary amines Greater stability 11.2 A Sulfhydryls SPDP Primaryamines Thiolation  6.8 A Sulfhydryls Cleavable cross-linking LC-SPDPPrimary ammes Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC- Primaryamines Extended spacer arm 15.6 A SPDP Sulfhydryls Water-soluble SMCCPrimary amines Stable maleimide reactive 11.6 A Sulfhydryls groupEnzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-Primary amines Stable maleimide reactive 11.6 A SMCC Sulfhydryls groupWater-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody conjugation  9.9 A Sulfhydryls Hapten-carrier proteinconjugation Sulfo- Primary amines Water-soluble  9.9 A MBS SulfhydrylsSIAB Primary amines Enzyme-antibody conjugation 10.6 A SulfhydrylsSulfo- Primary amines Water-soluble 10.6 A SIAB Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo- Primary amines Extended spacer arm 14.5 A SMPBSulfhydryls Water-soluble EDC/ Primary amines Hapten-Carrier conjugation 0 Sulfo- Carboxyl NHS groups ABH Carbohydrates Reacts with sugar groups11.9 A Nonselective

[0195] In instances where a particular polypeptide does not contain aresidue amenable for a given cross-linking reagent in its nativesequence, conservative genetic or synthetic amino acid changes in theprimary sequence can be utilized.

[0196] VII. Combination Treatments/Cancer Therapies

[0197] In order to increase the effectiveness of a chimeric polypeptideof the present invention, or expression construct coding therefor, itmay be desirable to combine these compositions with other agentseffective in the treatment of hyperproliferative disease, such asanti-cancer agents. A hyperproliferative disease includes diseases andconditions that are associated with any sort of abnormal cell growth orabnormal growth regulation. In methods of the present invention,preferably the patient is a human. A variety of hyperproliferativediseases can be treated according to the methods of the presentinvention. Some of the hyperproliferative diseases contemplated fortreatment in the present invention are psoriasis, rheumatoid arthritis(RA), inflammatory bowel disease (IBD), osteoarthritis (OA) andpre-neoplastic lesions in the mouth, prostate, breast, lung etc. Thepresent invention has important ramifications particularly with respectto one hyperproliferative disease: cancer.

[0198] Thus, in certain embodiments, the hyperproliferative disease isfurther defined as cancer. In still further embodiments, the cancer ismelanoma, non-small cell lung, small-cell lung, lung, hepatocarcinoma,retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia,neuroblastoma, head, neck, breast, pancreatic, prostate, renal, bone,testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma,brain, colon, sarcoma or bladder. The cancer may include a tumorcomprised of tumor cells. In other embodiments, the hyperproliferativedisease is rheumatoid arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions(such as adenomatous hyperplasia and prostatic intraepithelialneoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

[0199] An “anti-cancer” agent is capable of negatively affecting cancerin a subject, for example, by killing cancer cells, inducing apoptosisin cancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theexpression construct and the agent(s) or multiple factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

[0200] Tumor cell resistance to chemotherapy and radiotherapy agentsrepresents a major problem in clinical oncology. One goal of currentcancer research is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver, et al., 1992). In the context ofthe present invention, it is contemplated that chimeric polypepidescould be used similarly in conjunction with chemotherapeutic,radiotherapeutic, gene therapy, or immunotherapeutic intervention, inaddition to other pro-apoptotic or cell cycle regulating agents.

[0201] Alternatively, the therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone may contact the cell with both modalities within about 12-24 h ofeach other and, more preferably, within about 6-12 h of each other. Insome situations, it may be desirable to extend the time period fortreatment significantly, however, where several d (2, 3, 4, 5, 6 or 7)to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

[0202] Various combinations may be employed, gene therapy is “A” and thesecondary agent, such as radio- or chemotherapy, is “B”:

[0203] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/AB/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/AA/B/A/A A/A/B/A

[0204] Administration of the therapeutic expression constructs of thepresent invention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

[0205] A. Chemotherapy

[0206] Cancer therapies also include a variety of combination therapieswith both chemical and radiation based treatments. Combinationchemotherapies include, for example, cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

[0207] B. Radiotherapy

[0208] Other factors that cause DNA damage and have been usedextensively include what are commonly known as γ-rays, X-rays, and/orthe directed delivery of radioisotopes to tumor cells. Other forms ofDNA damaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

[0209] The terms “contacted” and “exposed,” when applied to a cell, areused herein to describe the process by which a therapeutic construct anda chemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

[0210] C. Immunotherapy

[0211] Immunotherapeutics, generally, rely on the use of immune effectorcells and molecules to target and destroy cancer cells. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually effect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

[0212] Immunotherapy, thus, could be used as part of a combined therapy,in conjunction with gene therapy. The general approach for combinedtherapy is discussed below. Generally, the tumor cell must bear somemarker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155.

[0213] D. Genes

[0214] In yet another embodiment, the secondary treatment is a genetherapy in which a therapeutic polynucleotide is administered before,after, or at the same time as a chimeric polypeptide of the presentinvention. Delivery of a chimeric polypeptide in conjuction with asecond vector encoding one of the following gene products will have acombined anti-hyperproliferative effect on target tissues.Alternatively, a single vector encoding both genes may be used. Avariety of proteins are encompassed within the invention, some of whichare described below.

[0215] 1. Inducers of Cellular Proliferation

[0216] The proteins that induce cellular proliferation further fall intovarious categories dependent on function. The commonality of all ofthese proteins is their ability to regulate cellular proliferation. Forexample, a form of PDGF, the sis oncogene, is a secreted growth factor.Oncogenes rarely arise from genes encoding growth factors, and at thepresent, sis is the only known naturally-occurring oncogenic growthfactor. In one embodiment of the present invention, it is contemplatedthat anti-sense mRNA directed to a particular inducer of cellularproliferation is used to prevent expression of the inducer of cellularproliferation.

[0217] The proteins FMS, ErbA, ErbB and neu are growth factor receptors.Mutations to these receptors result in loss of regulatable function. Forexample, a point mutation affecting the transmembrane domain of the Neureceptor protein results in the neu oncogene. The erbA oncogene isderived from the intracellular receptor for thyroid hormone. Themodified oncogenic ErbA receptor is believed to compete with theendogenous thyroid hormone receptor, causing uncontrolled growth.

[0218] The largest class of oncogenes includes the signal transducingproteins (e.g., Src, Abl and Ras). The protein Src is a cytoplasmicprotein-tyrosine kinase, and its transformation from proto-oncogene tooncogene in some cases, results via mutations at tyrosine residue 527.In contrast, transformation of GTPase protein ras from proto-oncogene tooncogene, in one example, results from a valine to glycine mutation atamino acid 12 in the sequence, reducing ras GTPase activity.

[0219] The proteins Jun, Fos and Myc are proteins that directly exerttheir effects on nuclear functions as transcription factors.

[0220] 2. Inhibitors of Cellular Proliferation

[0221] The tumor suppressor oncogenes function to inhibit excessivecellular proliferation. The inactivation of these genes destroys theirinhibitory activity, resulting in unregulated proliferation. The tumorsuppressors p53, p16 and C-CAM are described below.

[0222] High levels of mutant p53 have been found in many cellstransformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses. The p53 gene is a frequent target of mutationalinactivation in a wide variety of human tumors and is already documentedto be the most frequently mutated gene in common human cancers. It ismutated in over 50% of human NSCLC (Hollstein et al., 1991) and in awide spectrum of other tumors.

[0223] The p53 gene encodes a 393-amino acid phosphoprotein that canform complexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue

[0224] Wild-type p53 is recognized as an important growth regulator inmany cell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

[0225] Another inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16^(INK4) has been biochemically characterized as aprotein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p 16 also is known to regulatethe function of CDK6.

[0226] p16^(INK4) belongs to a newly described class of CDK-inhibitoryproteins that also includes p16^(B), p19, p21^(WAF1), and p27^(KIP1).The p16^(INK4) gene maps to 9p21, a chromosome region frequently deletedin many tumor types. Homozygous deletions and mutations of thep16^(INK4) gene are frequent in human tumor cell lines. This evidencesuggests that the p16^(INK4) gene is a tumor suppressor gene. Thisinterpretation has been challenged, however, by the observation that thefrequency of the p16^(INK4) gene alterations is much lower in primaryuncultured tumors than in cultured cell lines (Caldas et al., 1994;Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994; Kamb etal., 1994; Mori et al., 1994; Okamoto et al., 1994; Nobori et al., 1995;Orlow et al., 1994; Arap et al., 1995). Restoration of wild-typep16^(INK4) function by transfection with a plasmid expression vectorreduced colony formation by some human cancer cell lines (Okamoto, 1994;Arap, 1995).

[0227] Other genes that may be employed according to the presentinvention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1,p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genesinvolved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF,or their receptors) and MCC.

[0228] 3. Regulators of Programmed Cell Death

[0229] Apoptosis, or programmed cell death, is an essential process fornormal embryonic development, maintaining homeostasis in adult tissues,and suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Cleary et al., 1986; Tsujimoto et al, 1985; Tsujimoto andCroce, 1986). The evolutionarily conserved Bcl-2 protein now isrecognized to be a member of a family of related proteins, which can becategorized as death agonists or death antagonists.

[0230] Subsequent to its discovery, it was shown that Bcl-2 acts tosuppress cell death triggered by a variety of stimuli. Also, it now isapparent that there is a family of Bcl-2 cell death regulatory proteinswhich share in common structural and sequence homologies. Thesedifferent family members have been shown to either possess similarfunctions to Bcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1)or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak,Bik, Bim, Bid, Bad, Harakiri).

[0231] E. Surgery

[0232] Approximately 60% of persons with cancer will undergo surgery ofsome type, which includes preventative, diagnostic or staging, curativeand palliative surgery. Curative surgery is a cancer treatment that maybe used in conjunction with other therapies, such as the treatment ofthe present invention, chemotherapy, radiotherapy, hormonal therapy,gene therapy, immunotherapy and/or alternative therapies.

[0233] Curative surgery includes resection in which all or part ofcancerous tissue is physically removed, excised, and/or destroyed. Tumorresection refers to physical removal of at least part of a tumor. Inaddition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and miscopically controlledsurgery (Mohs' surgery). It is further contemplated that the presentinvention may be used in conjunction with removal of superficialcancers, precancers, or incidental amounts of normal tissue.

[0234] Upon excision of part of all of cancerous cells, tissue, ortumor, a cavity may be formed in the body. Treatment may be accomplishedby perfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

[0235] F. Other agents

[0236] It is contemplated that other agents may be used in combinationwith the present invention to improve the therapeutic efficacy oftreatment. These additional agents include immunomodulatory agents,agents that affect the upregulation of cell surface receptors and GAPjunctions, cytostatic and differentiation agents, inhibitors of celladehesion, or agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2and other cytokines; F42K and other cytokine analogs; or MIP-1,MIP-1beta, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5 /TRAIL would potentiate theapoptotic inducing abililties of the present invention by establishmentof an autocrine or paracrine effect on hyperproliferative cells.Increases intercellular signaling by elevating the number of GAPjunctions would increase the anti-hyperproliferative effects on theneighboring hyperproliferative cell population. In other embodiments,cytostatic or differentiation agents can be used in combination with thepresent invention to improve the anti-hyerproliferative efficacy of thetreatments. Inhibitors of cell adehesion are contemplated to improve theefficacy of the present invention. Examples of cell adhesion inhibitorsare focal adhesion kinase (FAKs) inhibitors and Lovastatin. It isfurther contemplated that other agents that increase the sensitivity ofa hyperproliferative cell to apoptosis, such as the antibody c225, couldbe used in combination with the present invention to improve thetreatment efficacy.

[0237] Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

[0238] VIII. Pharmaceutical Preparations

[0239] Pharmaceutical compositions of the present invention comprise aneffective amount of one or more chimeric polypeptides or chimericpolypeptides and at least one additional agent dissolved or dispersed ina pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one chimeric polypeptide or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

[0240] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

[0241] The chimeric polypeptide may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g. aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0242] The actual dosage amount of a composition of the presentinvention administered to an animal patient can be determined byphysical and physiological factors such as body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

[0243] In certain embodiments, pharmaceutical compositions may comprise,for example, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

[0244] In any case, the composition may comprise various antioxidants toretard oxidation of one or more component. Additionally, the preventionof the action of microorganisms can be brought about by preservativessuch as various antibacterial and antifungal agents, including but notlimited to parabens (e.g., methylparabens, propylparabens),chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0245] The chimeric polypeptide may be formulated into a composition ina free base, neutral or salt form. Pharmaceutically acceptable salts,include the acid addition salts, e.g., those formed with the free aminogroups of a proteinaceous composition, or which are formed withinorganic acids such as for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric or mandelic acid.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine.

[0246] In embodiments where the composition is in a liquid form, acarrier can be a solvent or dispersion medium comprising but not limitedto, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils,liposomes) and combinations thereof. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin; bythe maintenance of the required particle size by dispersion in carrierssuch as, for example liquid polyol or lipids; by the use of surfactantssuch as, for example hydroxypropylcellulose; or combinations thereofsuch methods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

[0247] In other embodiments, one may use eye drops, nasal solutions orsprays, aerosols or inhalants in the present invention. Suchcompositions are generally designed to be compatible with the targettissue type. In a non-limiting example, nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, in preferred embodiments the aqueous nasal solutionsusually are isotonic or slightly buffered to maintain a pH of about 5.5to about 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, drugs, or appropriate drug stabilizers,if required, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

[0248] In certain embodiments, the chimeric polypeptide is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

[0249] In certain preferred embodiments an oral composition may compriseone or more binders, excipients, disintegration agents, lubricants,flavoring agents, and combinations thereof. In certain embodiments, acomposition may comprise one or more of the following: a binder, suchas, for example, gum tragacanth, acacia, cornstarch, gelatin orcombinations thereof; an excipient, such as, for example, dicalciumphosphate, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate or combinations thereof; adisintegrating agent, such as, for example, corn starch, potato starch,alginic acid or combinations thereof; a lubricant, such as, for example,magnesium stearate; a sweetening agent, such as, for example, sucrose,lactose, saccharin or combinations thereof; a flavoring agent, such as,for example peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

[0250] Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

[0251] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

[0252] The composition must be stable under the conditions ofmanufacture and storage, and preserved against the contaminating actionof microorganisms, such as bacteria and fungi. It will be appreciatedthat endotoxin contamination should be kept minimally at a safe level,for example, less that 0.5 ng/mg protein.

[0253] In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

[0254] IX. Lipid Compositions

[0255] In certain embodiments, the present invention employs a novelcomposition comprising one or more lipids associated with at least onechimeric polypeptide. A lipid is a substance that is characteristicallyinsoluble in water and extractable with an organic solvent. Lipidsinclude, for example, the substances comprising the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds whichare well known to those of skill in the art which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

[0256] A lipid may be naturally occurring or synthetic (i.e., designedor produced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

[0257] A. Lipid Types

[0258] A neutral fat may comprise a glycerol and a fatty acid. A typicalglycerol is a three carbon alcohol. A fatty acid generally is a moleculecomprising a carbon chain with an acidic moeity (e.g., carboxylic acid)at an end of the chain. The carbon chain may of a fatty acid may be ofany length, however, it is preferred that the length of the carbon chainbe of from about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, 11, about 12, about 13, about 14, about 15, about16, about 17, about 18, about 19, about about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30 ormore carbon atoms, and any range derivable therein. However, a preferredrange is from about 14 to about 24 carbon atoms in the chain portion ofthe fatty acid, with about 16 to about 18 carbon atoms beingparticularly preferred in certain embodiments. In certain embodimentsthe fatty acid carbon chain may comprise an odd number of carbon atoms,however, an even number of carbon atoms in the chain may be preferred incertain embodiments. A fatty acid comprising only single bonds in itscarbon chain is called saturated, while a fatty acid comprising at leastone double bond in its chain is called unsaturated.

[0259] Specific fatty acids include, but are not limited to, linoleicacid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauricacid, myristic acid, arachidic acid, palmitoleic acid, arachidonic acidricinoleic acid, tuberculosteric acid, lactobacillic acid. An acidicgroup of one or more fatty acids is covalently bonded to one or morehydroxyl groups of a glycerol. Thus, a monoglyceride comprises aglycerol and one fatty acid, a diglyceride comprises a glycerol and twofatty acids, and a triglyceride comprises a glycerol and three fattyacids.

[0260] A phospholipid generally comprises either glycerol or ansphingosine moiety, an ionic phosphate group to produce an amphipathiccompound, and one or more fatty acids. Types of phospholipids include,for example, phophoglycerides, wherein a phosphate group is linked tothe first carbon of glycerol of a diglyceride, and sphingophospholipids(e.g., sphingomyelin), wherein a phosphate group is esterified to asphingosine amino alcohol. Another example of a sphingophospholipid is asulfatide, which comprises an ionic sulfate group that makes themolecule amphipathic. A phopholipid may, of course, comprise furtherchemical groups, such as for example, an alcohol attached to thephosphate group. Examples of such alcohol groups include serine,ethanolamine, choline, glycerol and inositol. Thus, specificphosphoglycerides include a phosphatidyl serine, a phosphatidylethanolamine, a phosphatidyl choline, a phosphatidyl glycerol or aphosphatidyl inositol. Other phospholipids include a phosphatidic acidor a diacetyl phosphate. In one aspect, a phosphatidylcholine comprisesa dioleoylphosphatidylcholine (a.k.a. cardiolipin), an eggphosphatidylcholine, a dipalmitoyl phosphalidycholine, a monomyristoylphosphatidylcholine, a monopalmitoyl phosphatidylcholine, a monostearoylphosphatidylcholine, a monooleoyl phosphatidylcholine, a dibutroylphosphatidylcholine, a divaleroyl phosphatidylcholine, a dicaproylphosphatidylcholine, a diheptanoyl phosphatidylcholine, a dicapryloylphosphatidylcholine or a distearoyl phosphatidylcholine.

[0261] A glycolipid is related to a sphinogophospholipid, but comprisesa carbohydrate group rather than a phosphate group attached to a primaryhydroxyl group of the sphingosine. A type of glycolipid called acerebroside comprises one sugar group (e.g., a glucose or galactose)attached to the primary hydroxyl group. Another example of a glycolipidis a ganglioside (e.g., a monosialoganglioside, a GM1), which comprisesabout 2, about 3, about 4, about 5, about 6, to about 7 or so sugargroups, that may be in a branched chain, attached to the primaryhydroxyl group. In other embodiments, the glycolipid is a ceramide(e.g., lactosylceramide).

[0262] A steroid is a four-membered ring system derivative of aphenanthrene. Steroids often possess regulatory functions in cells,tissues and organisms, and include, for example, hormones and relatedcompounds in the progestagen (e.g., progesterone), glucocoricoid (e.g.,cortisol), mineralocorticoid (e.g., aldosterone), androgen (e.g.,testosterone) and estrogen (e.g., estrone) families. Cholesterol isanother example of a steroid, and generally serves structural ratherthan regulatory functions. Vitamin D is another example of a sterol, andis involved in calcium absorption from the intestine.

[0263] A terpene is a lipid comprising one or more five carbon isoprenegroups. Terpenes have various biological functions, and include, forexample, vitamin A, coenyzme Q and carotenoids (e.g., lycopene andβ-carotene).

[0264] B. Charged and Neutral Lipid Compositions

[0265] In certain embodiments, a lipid component of a composition isuncharged or primarily uncharged. In one embodiment, a lipid componentof a composition comprises one or more neutral lipids. In anotheraspect, a lipid component of a composition may be substantially free ofanionic and cationic lipids, such as certain phospholipids (e.g.,phosphatidyl choline) and cholesterol. In certain aspects, a lipidcomponent of an uncharged or primarily uncharged lipid compositioncomprises about 95%, about 96%, about 97%, about 98%, about 99% or 100%lipids without a charge, substantially uncharged lipid(s), and/or alipid mixture with equal numbers of positive and negative charges.

[0266] In other aspects, a lipid composition may be charged. Forexample, charged phospholipids may be used for preparing a lipidcomposition according to the present invention and can carry a netpositive charge or a net negative charge. In a non-limiting example,diacetyl phosphate can be employed to confer a negative charge on thelipid composition, and stearylamine can be used to confer a positivecharge on the lipid composition.

[0267] C. Making Lipids

[0268] Lipids can be obtained from natural sources, commercial sourcesor chemically synthesized, as would be known to one of ordinary skill inthe art. For example, phospholipids can be from natural sources, such asegg or soybean phosphatidylcholine, brain phosphatidic acid, brain orplant phosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine. In another example, lipids suitable for useaccording to the present invention can be obtained from commercialsources. For example, dimyristyl phosphatidylcholine (“DMPC”) can beobtained from Sigma Chemical Co., dicetyl phosphate (“DCP”) is obtainedfrom K & K Laboratories (Plainview, N.Y.); cholesterol (“Chol”) isobtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol(“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc.(Birmingham, Ala.). In certain embodiments, stock solutions of lipids inchloroform or chloroform/methanol can be stored at about −20° C.Preferably, chloroform is used as the only solvent since it is morereadily evaporated than methanol.

[0269] D. Lipid Composition Structures

[0270] In a preferred embodiment of the invention, the chimericpolypeptide may be associated with a lipid. A chimeric polypeptideassociated with a lipid may be dispersed in a solution containing alipid, dissolved with a lipid, emulsified with a lipid, mixed with alipid, combined with a lipid, covalently bonded to a lipid, contained asa suspension in a lipid, contained or complexed with a micelle orliposome, or otherwise associated with a lipid or lipid structure. Alipid or lipid/chimeric polypeptide associated composition of thepresent invention is not limited to any particular structure. Forexample, they may also simply be interspersed in a solution, possiblyforming aggregates which are not uniform in either size or shape. Inanother example, they may be present in a bilayer structure, asmicelles, or with a “collapsed” structure. In another non-limitingexample, a lipofectamine (Gibco BRL)-chimeric polypeptide or Superfect(Qiagen)-chimeric polypeptide complex is also contemplated.

[0271] In certain embodiments, a lipid composition may comprise about1%, about 2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%,about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%,about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%,about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%,about 81%, about 82%, about 83%, about 84%, about about 85%, about 86%,about 87%, about 88%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type ornon-lipid component such as a drug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a drug. Thus, it is contemplatedthat lipid compositions of the present invention may comprise any of thelipids, lipid types or other components in any combination or percentagerange.

[0272] 1. Emulsions

[0273] A lipid may be comprised in an emulsion. A lipid emulsion is asubstantially permanent heterogenous liquid mixture of two or moreliquids that do not normally dissolve in each other, by mechanicalagitation or by small amounts of additional substances known asemulsifiers. Methods for preparing lipid emulsions and adding additionalcomponents are well known in the art (e.g., Modem Pharmaceutics, 1990,incorporated herein by reference).

[0274] For example, one or more lipids are added to ethanol orchloroform or any other suitable organic solvent and agitated by hand ormechanical techniques. The solvent is then evaporated from the mixtureleaving a dried glaze of lipid. The lipids are resuspended in aqueousmedia, such as phosphate buffered saline, resulting in an emulsion. Toachieve a more homogeneous size distribution of the emulsified lipids,the mixture may be sonicated using conventional sonication techniques,further emulsified using microfluidization (using, for example, aMicrofluidizer, Newton, Mass.), and/or extruded under high pressure(such as, for example, 600 psi) using an Extruder Device (LipexBiomembranes, Vancouver, Canada).

[0275] 2. Micelles

[0276] A lipid may be comprised in a micelle. A micelle is a cluster oraggregate of lipid compounds, generally in the form of a lipidmonolayer, and may be prepared using any micelle producing protocolknown to those of skill in the art (e.g., Canfield et al., 1990;El-Gorab et al, 1973; Colloidal Surfactant, 1963; and Catalysis inMicellar and Macromolecular Systems, 1975, each incorporated herein byreference). For example, one or more lipids are typically made into asuspension in an organic solvent, the solvent is evaporated, the lipidis resuspended in an aqueous medium, sonicated and then centrifuged.

[0277] 3. Liposomes

[0278] In particular embodiments, a lipid comprises a liposome. A“liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a bilayer membrane, generally comprising aphospholipid, and an inner medium that generally comprises an aqueouscomposition.

[0279] A multilamellar liposome has multiple lipid layers separated byaqueous medium. They form spontaneously when lipids comprisingphospholipids are suspended in an excess of aqueous solution. The lipidcomponents undergo self-rearrangement before the formation of closedstructures and entrap water and dissolved solutes between the lipidbilayers (Ghosh and Bachhawat, 1991). Lipophilic molecules or moleculeswith lipophilic regions may also dissolve in or associate with the lipidbilayer.

[0280] In certain less preferred embodiments, phospholipids from naturalsources, such as egg or soybean phosphatidylcholine, brain phosphatidicacid, brain or plant phosphatidylinositol, heart cardiolipin and plantor bacterial phosphatidylethanolamine are preferably not used as theprimary phosphatide, i.e., constituting 50% or more of the totalphosphatide composition or a liposome, because of the instability andleakiness of the resulting liposomes.

[0281] In particular embodiments, a lipid and/or chimeric polypeptidemay be, for example, encapsulated in the aqueous interior of a liposome,interspersed within the lipid bilayer of a liposome, attached to aliposome via a linking molecule that is associated with both theliposome and the chimeric polypeptide, entrapped in a liposome,complexed with a liposome, etc.

[0282] a. Making Liposomes

[0283] A liposome used according to the present invention can be made bydifferent methods, as would be known to one of ordinary skill in theart. Phospholipids can form a variety of structures other than liposomeswhen dispersed in water, depending on the molar ratio of lipid to water.At low ratios the liposome is the preferred structure.

[0284] For example, a phospholipid (Avanti Polar Lipids, Alabaster,Ala.), such as for example the neutral phospholipiddioleoylphosphatidylcholine (DOPC), is dissolved in tert-butanol. Thelipid(s) is then mixed with the chimeric polypeptide, and/or othercomponent(s). Tween 20 is added to the lipid mixture such that Tween 20is about 5% of the composition's weight. Excess tert-butanol is added tothis mixture such that the volume of tert-butanol is at least 95%. Themixture is vortexed, frozen in a dry ice/acetone bath and lyophilizedovernight. The lyophilized preparation is stored at −20° C. and can beused up to three months. When required the lyophilized liposomes arereconstituted in 0.9% saline. The average diameter of the particlesobtained using Tween 20 for encapsulating the chimeric polypeptide isabout 0.7 to about 1.0 μm in diameter.

[0285] Alternatively, a liposome can be prepared by mixing lipids in asolvent in a container, e.g., a glass, pear-shaped flask. The containershould have a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

[0286] Dried lipids can be hydrated at approximately 25-50 mMphospholipid in sterile, pyrogen-free water by shaking until all thelipid film is resuspended. The aqueous liposomes can be then separatedinto aliquots, each placed in a vial, lyophilized and sealed undervacuum.

[0287] In other alternative methods, liposomes can be prepared inaccordance with other known laboratory procedures (e.g., see Bangham etal., 1965; Gregoriadis, 1979; Deamer and Uster 1983, Szoka andPapahadjopoulos, 1978, each incorporated herein by reference in relevantpart). These methods differ in their respective abilities to entrapaqueous material and their respective aqueous space-to-lipid ratios.

[0288] The dried lipids or lyophilized liposomes prepared as describedabove may be dehydrated and reconstituted in a solution of inhibitorypeptide and diluted to an appropriate concentration with an suitablesolvent, e.g., DPBS. The mixture is then vigorously shaken in a vortexmixer. Unencapsulated additional materials, such as agents including butnot limited to hormones, drugs, nucleic acid constructs and the like,are removed by centrifugation at 29,000×g and the liposomal pelletswashed. The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

[0289] The size of a liposome varies depending on the method ofsynthesis. Liposomes in the present invention can be a variety of sizes.In certain embodiments, the liposomes are small, e.g., less than about100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less thanabout 50 nm in external diameter. In preparing such liposomes, anyprotocol described herein, or as would be known to one of ordinary skillin the art may be used. Additional non-limiting examples of preparingliposomes are described in U.S. Pat. Nos. 4,728,578, 4,728,5754,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; InternationalApplications PCT/US85/01161 and PCT/US89/05040; U.K. Patent ApplicationGB 2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al. 1987;Mayhew et al., 1984; Cheng et al., 1987; and Liposome Technology, 1984,each incorporated herein by reference).

[0290] A liposome suspended in an aqueous solution is generally in theshape of a spherical vesicle, having one or more concentric layers oflipid bilayer molecules. Each layer consists of a parallel array ofmolecules represented by the formula XY, wherein X is a hydrophilicmoiety and Y is a hydrophobic moiety. In aqueous suspension, theconcentric layers are arranged such that the hydrophilic moieties tendto remain in contact with an aqueous phase and the hydrophobic regionstend to self-associate. For example, when aqueous phases are presentboth within and without the liposome, the lipid molecules may form abilayer, known as a lamella, of the arrangement XY-YX. Aggregates oflipids may form when the hydrophilic and hydrophobic parts of more thanone lipid molecule become associated with each other. The size and shapeof these aggregates will depend upon many different variables, such asthe nature of the solvent and the presence of other compounds in thesolution.

[0291] The production of lipid formulations often is accomplished bysonication or serial extrusion of liposomal mixtures after (I) reversephase evaporation (II) dehydration-rehydration (III) detergent dialysisand (IV) thin film hydration. In one aspect, a contemplated method forpreparing liposomes in certain embodiments is heating sonicating, andsequential extrusion of the lipids through filters or membranes ofdecreasing pore size, thereby resulting in the formation of small,stable liposome structures. This preparation produces liposomal/chimericpolypeptide or liposomes only of appropriate and uniform size, which arestructurally stable and produce maximal activity. Such techniques arewell-known to those of skill in the art (see, for example Lang et al.,1990).

[0292] Once manufactured, lipid structures can be used to encapsulatecompounds that are toxic (e.g., chemotherapeutics) or labile (e.g.,nucleic acids) when in circulation. The physical characteristics ofliposomes depend on pH, ionic strength and/or the presence of divalentcations. Liposomes can show low permeability to ionic and/or polarsubstances, but at elevated temperatures undergo a phase transitionwhich markedly alters their permeability. The phase transition involvesa change from a closely packed, ordered structure, known as the gelstate, to a loosely packed, less-ordered structure, known as the fluidstate. This occurs at a characteristic phase-transition temperatureand/or results in an increase in permeability to ions, sugars and/ordrugs. Liposomal encapsulation has resulted in a lower toxicity and alonger serum half-life for such compounds (Gabizon et al., 1990).

[0293] Liposomes interact with cells to deliver agents via fourdifferent mechanisms: Endocytosis by phagocytic cells of thereticuloendothelial system such as macrophages and/or neutrophils;adsorption to the cell surface, either by nonspecific weak hydrophobicand/or electrostatic forces, and/or by specific interactions withcell-surface components; fusion with the plasma cell membrane byinsertion of the lipid bilayer of the liposome into the plasma membrane,with simultaneous release of liposomal contents into the cytoplasm;and/or by transfer of liposomal lipids to cellular and/or subcellularmembranes, and/or vice versa, without any association of the liposomecontents. Varying the liposome formulation can alter which mechanism isoperative, although more than one may operate at the same time.

[0294] Numerous disease treatments are using lipid based gene transferstrategies to enhance conventional or establish novel therapies, inparticular therapies for treating hyperproliferative diseases. Advancesin liposome formulations have improved the efficiency of gene transferin vivo (Templeton et al., 1997) and it is contemplated that liposomesare prepared by these methods. Alternate methods of preparinglipid-based formulations for nucleic acid delivery are described (WO99/18933).

[0295] In another liposome formulation, an amphipathic vehicle called asolvent dilution microcarrier (SDMC) enables integration of particularmolecules into the bi-layer of the lipid vehicle (U.S. Pat. No.5,879,703). The SDMCs can be used to deliver lipopolysaccharides,polypeptides, nucleic acids and the like. Of course, any other methodsof liposome preparation can be used by the skilled artisan to obtain adesired liposome formulation in the present invention.

[0296] b. Targeting Ligands

[0297] The targeting ligand can be either anchored in the hydrophobicportion of the complex or attached to reactive terminal groups of thehydrophilic portion of the complex. The targeting ligand can be attachedto the liposome via a linkage to a reactive group, e.g., on the distalend of the hydrophilic polymer. Preferred reactive groups include aminogroups, carboxylic groups, hydrazide groups, and thiol groups. Thecoupling of the targeting ligand to the hydrophilic polymer can beperformed by standard methods of organic chemistry that are known tothose skilled in the art. In certain embodiments, the totalconcentration of the targeting ligand can be from about 0.01 to about10% mol.

[0298] Targeting ligands are any ligand specific for a characteristiccomponent of the targeted region. Preferred targeting ligands includeproteins such as polyclonal or monoclonal antibodies, antibodyfragments, or chimeric antibodies, enzymes, or hormones, or sugars suchas mono-, oligo- and poly-saccharides (see, Heath et al., Chem. Phys.Lipids 40:347 (1986)) For example, disialoganglioside GD2 is a tumorantigen that has been identified neuroectodermal origin tumors, such asneuroblastoma, melanoma, small-cell lung carcinoma, glioma and certainsarcomas (Mujoo et al., 1986, Schulz et al., 1984). Liposomes containinganti-disialoganglioside GD2 monoclonal antibodies have been used to aidthe targeting of the liposomes to cells expressing the tumor antigen(Montaldo et al., 1999; Pagan et al., 1999). In another non-limitingexample, breast and gynecological cancer antigen specific antibodies aredescribed in U.S. Pat. No. 5,939,277, incorporated herein by reference.In a further non-limiting example, prostate cancer specific antibodiesare disclosed in U.S. Pat. No. 6,107,090, incorporated herein byreference. Thus, it is contemplated that the antibodies described hereinor as would be known to one of ordinary skill in the art may be used totarget specific tissues and cell types in combination with thecompositions and methods of the present invention. In certainembodiments of the invention, contemplated targeting ligands interactwith integrins, proteoglycans, glycoproteins, receptors or transporters.Suitable ligands include any that are specific for cells of the targetorgan, or for structures of the target organ exposed to the circulationas a result of local pathology, such as tumors.

[0299] In certain embodiments of the present invention, in order toenhance the transduction of cells, to increase transduction of targetcells, or to limit transduction of undesired cells, antibody or cyclicpeptide targeting moieties (ligands) are associated with the lipidcomplex. Such methods are known in the art. For example, liposomes havebeen described further that specifically target cells of the mammaliancentral nervous system (U.S. Pat. No. 5,786,214, incorporated herein byreference). The liposomes are composed essentially ofN-glutarylphosphatidylethanolamine, cholesterol and oleic acid, whereina monoclonal antibody specific for neuroglia is conjugated to theliposomes. It is contemplated that a monoclonal antibody or antibodyfragment may be used to target delivery to specific cells, tissues, ororgans in the animal, such as for example, brain, heart, lung, liver,etc.

[0300] Still further, a chimeric polypeptide may be delivered to atarget cell via receptor-mediated delivery and/or targeting vehiclescomprising a lipid or liposome. These take advantage of the selectiveuptake of macromolecules by receptor-mediated endocytosis that will beoccurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

[0301] Thus, in certain aspects of the present invention, a ligand willbe chosen to correspond to a receptor specifically expressed on thetarget cell population. A cell-specific chimeric polypeptide deliveryand/or targeting vehicle may comprise a specific binding ligand incombination with a liposome. The chimeric polypeptide to be deliveredare housed within a liposome and the specific binding ligand isfunctionally incorporated into a liposome membrane. The liposome willthus specifically bind to the receptor(s) of a target cell and deliverthe contents to a cell. Such systems have been shown to be functionalusing systems in which, for example, epidermal growth factor (EGF) isused in the receptor-mediated delivery of a nucleic acid to cells thatexhibit upregulation of the EGF receptor.

[0302] In certain embodiments, a receptor-mediated delivery and/ortargeting vehicles comprise a cell receptor-specific ligand and achimeric polypeptide-binding agent. Others comprise a cellreceptor-specific ligand to which chimeric polypeptide to be deliveredhas been operatively attached. For example, several ligands have beenused for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner etal., 1990 Perales et al., 1994; Myers, EPO 0273085), which establishesthe operability of the technique. In another example, specific deliveryin the context of another mammalian cell type has been described (Wu andWu, 1993; incorporated herein by reference).

[0303] In still further embodiments, the specific binding ligand maycomprise one or more lipids or glycoproteins that direct cell-specificbinding. For example, lactosyl-ceramide, a galactose-terminalasialganglioside, have been incorporated into liposomes and observed anincrease in the uptake of the insulin gene by hepatocytes (Nicolau etal., 1987). The asialoglycoprotein, asialofetuin, which containsterminal galactosyl residues, also has been demonstrated to targetliposomes to the liver (Spanjer and Scherphof, 1983; Hara et al., 1996).The sugars mannosyl, fucosyl or N-acetyl glucosamine, when coupled tothe backbone of a polypeptide, bind the high affinity manose receptor(U.S. Pat. No. 5,432,260, specifically incorporated herein by referencein its entirety). It is contemplated that the cell or tissue-specifictransforming constructs of the present invention can be specificallydelivered into a target cell or tissue in a similar manner.

[0304] In another example, lactosyl ceramide, and peptides that targetthe LDL receptor related proteins, such as apolipoprotein E3 (“Apo E”)have been useful in targeting liposomes to the liver (Spanjer andScherphof, 1983; WO 98/0748).

[0305] Folate and the folate receptor have also been described as usefulfor cellular targeting (U.S. Pat. No. 5,871,727). In this example, thevitamin folate is coupled to the complex. The folate receptor has highaffinity for its ligand and is overexpressed on the surface of severalmalignant cell lines, including lung, breast and brain tumors.Anti-folate such as methotrexate may also be used as targeting ligands.Transferrin mediated delivery systems target a wide range of replicatingcells that express the transferrin receptor (Gilliland et al., 1980).

[0306] C. Liposome/Nucleic Acid Combinations

[0307] In certain embodiments, a liposome/chimeric polypeptide maycomprise a nucleic acid, such as, for example, an oligonucleotide, apolynucleotide or a nucleic acid construct (e.g., an expression vector).Where a bacterial promoter is employed in the DNA construct that is tobe transfected into eukaryotic cells, it also will be desirable toinclude within the liposome an appropriate bacterial polymerase.

[0308] It is contemplated that when the liposome/chimeric polypeptidecomposition comprises a cell or tissue specific nucleic acid, thistechnique may have applicability in the present invention. In certainembodiments, lipid-based non-viral formulations provide an alternativeto viral gene therapies. Although many cell culture studies havedocumented lipid-based non-viral gene transfer, systemic gene deliveryvia lipid-based formulations has been limited. A major limitation ofnon-viral lipid-based gene delivery is the toxicity of the cationiclipids that comprise the non-viral delivery vehicle. The in vivotoxicity of liposomes partially explains the discrepancy between invitro and in vivo gene transfer results. Another factor contributing tothis contradictory data is the difference in liposome stability in thepresence and absence of serum proteins. The interaction betweenliposomes and serum proteins has a dramatic impact on the stabilitycharacteristics of liposomes (Yang and Huang, 1997). Cationic liposomesattract and bind negatively charged serum proteins. Liposomes coated byserum proteins are either dissolved or taken up by macrophages leadingto their removal from circulation. Current in vivo liposomal deliverymethods use aerosolization, subcutaneous, intradermal, intratumoral, orintracranial injection to avoid the toxicity and stability problemsassociated with cationic lipids in the circulation. The interaction ofliposomes and plasma proteins is largely responsible for the disparitybetween the efficiency of in vitro (Felgner et al., 1987) and in vivogene transfer (Zhu et al., 1993; Philip et al., 1993; Solodin et al.,1995; Liu et al., 1995; Thierry et al., 1995; Tsukamoto et al., 1995;Aksentijevich et al., 1996).

[0309] An exemplary method for targeting viral particles to cells thatlack a single cell-specific marker has been described (U.S. Pat. No.5,849,718). In this method, for example, antibody A may have specificityfor tumor, but also for normal heart and lung tissue, while antibody Bhas specificity for tumor but also normal liver cells. The use ofantibody A or antibody B alone to deliver an anti-proliferative nucleicacid to the tumor would possibly result in unwanted damage to heart andlung or liver cells. However, antibody A and antibody B can be usedtogether for improved cell targeting. Thus, antibody A is coupled to agene encoding an anti-proliferative nucleic acid and is delivered, via areceptor mediated uptake system, to tumor as well as heart and lungtissue. However, the gene is not transcribed in these cells as they lacka necessary transcription factor. Antibody B is coupled to a universallyactive gene encoding the transcription factor necessary for thetranscription of the anti-proliferative nucleic acid and is delivered totumor and liver cells. Therefore, in heart and lung cells only theinactive anti-proliferative nucleic acid is delivered, where it is nottranscribed, leading to no adverse effects. In liver cells, the geneencoding the transcription factor is delivered and transcribed, but hasno effect because no an anti-proliferative nucleic acid gene is present.In tumor cells, however, both genes are delivered and the transcriptionfactor can activate transcription of the anti-proliferative nucleicacid, leading to tumor-specific toxic effects.

[0310] The addition of targeting ligands for gene delivery for thetreatment of hyperproliferative diseases permits the delivery of geneswhose gene products are more toxic than do non-targeted systems.Examples of the more toxic genes that can be delivered includespro-apoptotic genes such as Bax and Bak plus genes derived from virusesand other pathogens such as the adenoviral E4orf4 and the E.coli purinenucleoside phosphorylase, a so-called “suicide gene” which converts theprodrug 6-methylpurine deoxyriboside to toxic purine 6-methylpurine.Other examples of suicide genes used with prodrug therapy are the E.coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0311] It is also possible to utilize untargeted or targeted lipidcomplexes to generate recombinant or modified viruses in vivo. Forexample, two or more plasmids could be used to introduce retroviralsequences plus a therapeutic gene into a hyperproliferative cell.Retroviral proteins provided in trans from one of the plasmids wouldpermit packaging of the second, therapeutic gene-carrying plasmid.Transduced cells, therefore, would become a site for production ofnon-replicative retroviruses carrying the therapeutic gene. Theseretroviruses would then be capable of infecting nearby cells. Thepromoter for the therapeutic gene may or may not be inducible or tissuespecific.

[0312] Similarly, the transferred nucleic acid may represent the DNA fora replication competent or conditionally replicating viral genome, suchas an adenoviral genome that lacks all or part of the adenoviral E1a orE2b region or that has one or more tissue-specific or induciblepromoters driving transcription from the E1a and/or E1b regions. Thisreplicating or conditional replicating nucleic acid may or may notcontain an additional therapeutic gene such as a tumor suppressor geneor anti-oncogene.

[0313] d. Lipid Administration

[0314] The actual dosage amount of a lipid composition (e.g., aliposome-chimeric polypeptide) administered to a patient can bedetermined by physical and physiological factors such as body weight,severity of condition, idiopathy of the patient and on the route ofadministration. With these considerations in mind, the dosage of a lipidcomposition for a particular subject and/or course of treatment canreadily be determined.

[0315] The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,rectally,topically, intratumorally, intramuscularly, intraperitoneally,subcutaneously, intravesicularlly, mucosally, intrapericardially,orally, topically, locally and/or using aerosol, injection, infusion,continuous infusion, localized perfusion bathing target cells directlyor via a catheter and/or lavage.

[0316] X. Antibody Preparation

[0317] A. Polyclonal Antibodies

[0318] Polyclonal antibodies are useful in the present inventionregarding multiple embodiments for the chimeric polypeptides. Polyclonalantibodies to the chimeric polypeptides generally are raised in animalsby multiple subcutaneous (sc) or intraperitoneal (ip) injections of thechimeric polypeptide and an adjuvant. It may be useful to conjugate thechimeric polypeptides or a fragment containing the target amino acidsequence to a protein that is immunogenic in the species to beimmunized, e.g. keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glytaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are different alkyl groups.

[0319] Animals are immunized against the immunogenic conjugates orderivatives by combining 1 mg of 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to {fraction (1/10)} the original amount ofconjugate in Freud's complete adjuvant by subcutaneous injection atmultiple sites. 7 to 14 days later the animals are bled and the serum isassayed for anti-chimeric polypeptides antibody titer. Animals areboosted until the titer plateaus. Preferably, the animal boosted withthe conjugate of the same chimeric polypeptides, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are used to enhance theimmune response.

[0320] B. Monoclonal antibodies

[0321] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Thus, the modifier “monoclonal” indicates the character of the antibodyas not being a mixture of discrete antibodies.

[0322] For example, the anti-chimeric polypeptide monoclonal antibodiesof the invention may be made using the hybridoma method first describedby Kohler and Milstein (1975), or may be made by recombinant DNA methods[Cabilly et al., U.S. Pat. No. 4,816,567].

[0323] In the hybridoma method, a mouse or other appropriate hostanimal, such as hamster is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)].

[0324] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0325] Preferred myeloma cells are those that fuse efficiently, supportstable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 cells available from the American Type CultureCollection, Rockville, Md. USA.

[0326] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against chimericpolypeptides. Preferably, the binding specificity of monoclonalantibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0327] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson and Pollard (1980).

[0328] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

[0329] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0330] DNA encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells of the invention serve as a preferred source of suchDNA. Once isolated, the DNA may be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequences(Morrison et al., 1984), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-chimeric polypeptide monoclonal antibody herein.

[0331] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a chimericpolypeptide and another antigen-combining site having specificity for adifferent antigen.

[0332] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0333] For diagnostic applications, the antibodies of the inventiontypically will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

[0334] Any method known in the art for separately conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter et al., (1962); David et al. (1974); Pain etal. (1981); and Nygren (1982).

[0335] The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,Inc., 1987).

[0336] Competitive binding assays rely on the ability of a labeledstandard (which may be a chimeric polypeptide or an immunologicallyreactive portion thereof) to compete with the test sample analyte(chimeric polypeptides) for binding with a limited amount of antibody.The amount of chimeric polypeptides in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0337] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three part complex (U.S. Pat. No. 4,376,110). The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

[0338] C. Humanized Antibodies

[0339] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al. (1986); Riechmann et al. (1988); Verhoeyen et al. (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0340] It is important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.For further details see U.S. application Ser. No. 07/934,373 filed Aug.21, 1992, which is a continuation-in-part of application Ser. No.07/715,272 filed Jun. 14, 1991.

[0341] D. Human antibodies

[0342] Human monoclonal antibodies can be made by the hybridoma method.Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor (1984), and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

[0343] It is now possible to produce transgenic animals (e.g. mice) thatare capable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.(1993).

[0344] Alternatively, the phage display technology (McCafferty et al.(1990) can be used to produce human antibodies and antibody fragments invitro, from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.

[0345] Because the filamentous particle contains a single-stranded DNAcopy of the phage genome, selections based on the functional propertiesof the antibody also result in selection of the gene encoding theantibody exhibiting those properties. Thus, the phage mimicks some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson and Chiswell(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al. (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al. (1991),or Griffith et al. (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., 1992). In this method, the affinity of “primary” humanantibodies obtained by phage display can be improved by sequentiallyreplacing the heavy and light chain V region genes with repertoires ofnaturally occurring variants (repertoires) of V domain genes obtainedfrom unimmunized donors. This techniques allows the production ofantibodies and antibody fragments with affinities in the nM range. Astrategy for making very large phage antibody repertoires (also known as“the mother-of-all libraries”) has been described by Waterhouse et al.(1993), and the isolation of a high affinity human antibody directlyfrom such large phage library is reported by Griffith et al. (1994).Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published Apr. 1, 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

[0346] E. Bispecific Antibodies

[0347] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for a chimeric polypeptide, the other one is for anyother antigen, and preferably for another receptor or receptor subunit.For example, bispecific antibodies specifically binding a chimericpolypeptide and neurotrophic factor, or two different chimericpolypeptides are within the scope of the present invention.

[0348] F. Methods for making Bispecific Antibodies are known in the art

[0349] Traditionally, the recombinant production of bispecificantibodies is based on the coexpression of two immunoglobulin heavychain-light chain pairs, where the two heavy chains have differentspecificities (Milstein and Cuello (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule, which is usually done by affinitychromatography steps, is rather cumbersome, and the product yields arelow. Similar procedures are disclosed in PCT application publication No.WO 93/08829 (published May 13, 1993), and in Traunecker et al. (1991).

[0350] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH₂ andCH₃ regions. It is preferred to have the first heavy chain constantregion (CH1) containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed incopending application Ser. No. 07/931,811 filed Aug. 17, 1992.

[0351] For further details of generating bispecific antibodies see, forexample, Suresh et al. (1986).

[0352] G. Heteroconjugate Antibodies

[0353] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

EXAMPLES

[0354] The following is an illustration of preferred embodiments forpracticing the present invention. However, they are not limitingexamples. Other examples and methods are possible in practicing thepresent invention.

Example 1 Materials

[0355] The following materials were utilized for multiple Examplesdescribed herein. The PCR reagents were obtained from Fisher Scientific(Pittsburgh, Pa.), and the molecular biology enzymes were purchased fromBoehringer Mannheim (Indianapolis, Ind.) or New England Biolabs(Beverly, Mass.). Bacterial strains, pET bacterial expression plasmidsand recombinant enterokinase were obtained from Novagen (Madison, Wis.).All other chemicals were from Sigma Chemical Company (St. Louis, Mo.) orFisher Scientific (Pittsburg, Pa.). Metal affinity resin (Talon orNichel agrose) was obtained from Clontech Laboratories (Palo Alto,Calif.). Tissue culture reagents were from Gibco BRL (Gaithersburg,Md.).

[0356] Human cutaneous T cell lymphoma (Hut78) from American TypeCulture Collection (ATCC, Manassas, Va.) cultured in RPMI 1640 mediumcontaining 10% fetal bovine serum (FBS). Porcine aortic endothelialcells transfected with either flt-1 receptor (PAE-Flt-1) or theflk-1/KDR receptor (PAE-Flk-1) were a gift from Dr. J. Waltenberger andcultured in F12 Nutrient Mixture (HAM) with 10% FBS. The human melanomaA375M cell-line was obtained from Dr. I. J. Fidler of the University ofTexas MD Anderson Cancer Center (Houston, Tex.) and were cultured in MEMsupplemented with 10% FBS.

Example 2 Methods—Cloning Human Granzyme B Gene

[0357] The following methods were utilized at least for Example 13.Hut78 RNA was isolated using GlassMAX RNA Microisolation Spin CartridgeSystem (Gibco BRL), and the quanitity of total RNA was then determined.Genomic DNA was then removed by incubating the sample with DNase I for15 min at room temperature. The DNase I was inactivated by adding EDTAsolution heating for 15 min at 65° C. The SUPERSCRIPT First-StrandSynthesis System for RT-PCR (Gibco BRL) was used to synthesize thefirst-strand with oligo (dT). The target cDNA (pre-mature human GranzymeB cDNA) was amplified using the primers: NcoIgb (5′ to 3′):GGTGGCGGTGGCTCCATGGAACCAATCCTGCTTCTG (SEQ ID NO:1) and CxhoIgb (5′ to3′): GCCACCGCCTCCCTCGAGCTATTAGTAGCGTTTCATGGT (SEQ ID NO:2) by PCR. PCRconditions included denaturation at 95° C. for 5 min, PCR cycle of 94°C. for 1 min, 50° C. for 1 min, 72° C. for 1 min, for a total of 30cycles; extension step was 72° C. for 5 min. A 1% agarose gel was run toconfirm the PCR product. The PCR product was then cloned into PCR 2.1 TAvector (Invitrogen; Carlsbad, Calif.) and designated gbTA. The gbTA wastransformed into INVαF′ competent cells, and the positive clones werescreened by blue/white colony screening or by PCR methods. The DNAs forpositive clones were isolated by using QIAprep Spin prep kit (Qiagen;Valencia, Calif.) and sequenced to confirm human granzyme B gene; thecorrect clone was identified as gbTA-2 (clone #2).

Example 3 Methods—Construction of Granzyme B-Vegf121 or GranzymeB-Scfvmel Fusion Genes

[0358] The following fusion constructs were utilized in multipleExamples described herein. The fusion construct Granzyme B-vegf121 wasan Ek-Granzyme B-G4S linker-Vegf121 format. The construction was basedon over-lap PCR method. Briefly, granzyme B coding sequence wasamplified from gbTA-2 by PCR using the primers: NgbEK (5′ to 3′):GGTACCGACGACGACGACAAGATCATCGGGGGACATGAG, Cgb (5′ to 3′) (SEQ ID NO:3)and GGAGCCACCGCCACCGTAGCGTTTCATGGT (SEQ ID NO:4). These were designed todelete the signal sequence of pre-mature granzyme B and insert anenterokinase cleavage site at the N-terminus, in addition to adding aG4S linker sequence to the C-terminus in order to link to vegf121 gene.Vegf121 sequence was amplified from a plasmid pET22-vegf121 (a gift fromDr. Phil Thorpe's group, the University of Texas Southwest MedicalSchool, Dallas, Tex.) by PCR using primers: Nvegf (5′ to 3′)GGTGGCGGTGGCTCCGCACCCATGGCAGAA (SEQ ID NO:5) and Cxhol veg (5′ to 3′)AAGGCTCGTGTCGACCTCGAGTCATTACCGCCTCGGCTTGTC (SEQ ID NO:6). ScFvMELsequence was amplified from a plasmid pET32-scFvMEL/TNF by PCR usingprimers: Nzme2 (5′ to 3′)GGTGGCGGTGGCTCCACGGACATTGTGATGACCCAGTCTCAAAAATTC (SEQ ID NO:7) and Czme2(5′ to 3′) GGAGCCACCGCCACCCTCGAGCTATCATGAGGAGACGGTGAGAGTGGT (SEQ IDNO:8). These primers added G4S linker sequence to the N-terminus tooverlap PCR link to the C-terminus of granzyme B, and a Xho I site wasincorporated at the C-terminus to facilitate subsequent cloning steps.Two stop codons were added at the C-terminus just before the Xho I site.The fused genes were linked together by the second PCR using primersNgbEK and Cxholveg (for granzyme B-vegf121) or NgbEK and Czme2 (forgranzyme B-scFvMEL). In order to clone the fused genes into pET32a (+)vector with an enterokinase site at the N-terminus of granzyme B, thefragment from pET32a (+) was amplified by PCR using primers T7 promoter(5′ to 3′) TAATACGACTCACTATAG (SEQ ID NO:9) and CpET32EK (5′ to 3′)CTTGTCGTCGTCGTCGGTACCCAGATCTGG (SEQ ID NO:10). The primer has anenterokinase site at the C-terminus overlapping with the N-terminus offused gene. By overlap PCR, the fusion genes EK-Granzyme B-vegf121 wereconstructed using primers T7 promoter and CxhoIveg, and the fusion genesEK-Granzyme B-scFvMEL were constructed using primers T7 promoter andCzme2. The PCR reactions were performed by thirty cycles of 94° C. for 1min, 50° C. for 1 min and 72° C. for 1 min, with an extension reactionat 72° C. for 5 min. Amplified fragments were separated by 1% agarosegel electrophoresis and purified by PCR purification kit (Qiagen). Thepurified PCR products were digested with Xba I and Xho I at 37° C. for 3hrs and then separated by 1% agarose gel electrophoresis, purified fromthe gels and cloned into pET32a (+) vector, designated pET32GrB-vegf121or pET32GrB-scFvMEL. The ligation mixture was transformed into DH5αcompetent cells, the positive clones were screened by PCR, and thensequenced. A clone having the T7 promoter, lac operator, rbs, Trx.tag,His.tag, S-tag, and enterokinase sites to granzyme B-G4S-vegf121 orgranzyme B-G4S-scFvMEL, with no second site mutations, was chosen fortransformation into AD494 (DE3)pLysS competent cells for furtherinduction and expression.

Example 4 Method—Induction and Expression of Granzyme B-Vegf121 orGranzyme B-Scfvmel Fusion Proteins in E. coli

[0359] The fusion constructs of Example 3 were induced and expressed asdescribed herein for utilization in multiple Examples elsewhere herein,including at least Examples 17 and 18. Bacterial colonies transformedwith the constructed plasmid were grown in Luria Broth (LB) growth mediacontaining 200 μg/ml ampicillin, 70 μg/ml chloramphenicol, and 15 μg/mlkanamycin, at 37° C. overnight at 240 rpm shaking. The cultures werethen diluted 1:100 in fresh LB media plus antibiotics and grown to A₆₀₀of 0.5 at 37° C. Thereafter, the cultures were induced by addition ofIPTG to a final concentration of 0.25 mM at 37° C. for 1.5 hrs. Thecells were harvested and resuspended in 10 mM Tris (pH 8.0) and storedfrozen at −80° C. for later purification.

Example 5 Methods—Purification of Granzyme B-vegf121 or GranzymeB-Scfvmel Fusion Protein

[0360] The fusion constructs induced and expressed in Example 4 werepurified as described herein for utilization in multiple Exampleselsewhere herein, including at least Examples 17 and 18. Theresuspension culture was lysed by addition of lysozyme to a finalconcentration of 100 μg/ml with agitation for 30 min at 4° C., which wasfollowed by sonication. Extracts were centrifuged at 10,800 g for 30min, and the supernatant was further centrifuged at 40,000 rpm for 1 hr.The supernatant containing only soluble protein was adjusted to 40 mMTris, pH 8.0, 10 mM imidazole and applied to a nickel-NTA agaroseequilibrated with the same buffer. After washing the nickel-NTA agarosewith 500 mM NaCl and 20 mM imidazole, the bound proteins were elutedwith 500 mM NaCl, and 500 mM imidazole. Absorbance (280 nM) and SDS-PAGEanalyses were performed to identify the polyhistidine-tagged protein,designated Pro-granzyme B-vegf121 or Pro-granzyme B-scFvMEL,respectively. The eluted pro-granzymeB-vegf121 or pro-granzymeB-scFvMELprotein was dialyzed against 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl.Progranzyme B moiety of granzyme B-vegf121 or granzyme B-scFvMEL wasactivated by the addition of recombinant bovine enterokinase (rEK) toremove the polyhistidine-tag according to the manufacturer's instruction(1 unit of rEK cleavage 50 μg protein, incubated at room temperature for16 hrs). The rEK was removed by EK capture agarose. The final proteinwas analyzed by SDS-PAGE and stored at 4° C.

Example 6 Methods—SDS-page and Western Blot Analysis

[0361] The following methods were performed for experiments as describedin, for example, Example 15. Protein samples were analyzed byelectrophoresis on an 8.5% SDS-PAGE under reducing conditions. The gelswere stained with coomassie blue. For western blotting analysis,proteins were transferred from gels into nitrocellulose membranes. Themembranes were blocked with 5% non-fat milk and incubated for 1 hr atroom temperature with mouse anti-granzyme B monoclonal antibody (1.0μg/ml) or mouse anti-vegf121 polyclonal antibody (1:2000 dilution) orrabbit anti-scFvzme polyclonal antibody (1:2000 dilution). Afterwashing, the membranes were incubated with goat anti-mouse/horseradishperoxidase conjugate (HRP-GAM, 1:5000 dilution) or goatanti-rabbit/horseradish peroxidase conjugate (HRP-GAR, 1:5000 dilution).After further washing, the membrane was developed using the Amersham(Piscataway, N.J.) ECL detection system and exposed to X-ray film.

Example 7 Methods—Enzyme Assays

[0362] The enzymatic activity of granzyme B was determined in acontinuous calorimetric assay, with BAADT(N-α-t-butoxycarbonyl-L-alanyl-L-alanyl-L-aspartyl-thiobenzyl ester) assubstrate. Assays were performed in 200 μl and consisted of enzyme in100 mM HEPES, pH7.5, 10 mM CaCl₂, 1 mM 5,5′-dithiobis-2-nitrobenzoicacid, 0.2 mM substrate at 25° C. The change in absorbance at OD₄₀₅ wasmeasured on a Thermomax plate reader. Absorbance increases wereconverted to enzymatic rates by using an extinction coefficient of 13,100 cm-1M-1 that differed from the usual extinction coefficient of 13,600 cm-1M-1 at 412 nm reported by Ellman.

Example 8 Methods—Detection of Scfvmel Moiety of Granzyme B-Scfvmel

[0363] Reacti-BindTM Protein L Coated Plates from PIERCE (Rockford,Ill.) were used for detection of scFvMEL moiety of GranzymeB-scFvMEL,based on ELISA method. Briefly, pre-coated Protein L was blocked by 5%BSA, and the reaction was purified with Granzyme B-scFvMEL or otherscFvMEL fusion proteins at various concentrations, respectively. Afterwashing, the proteins were incubated with rabbit ant-scFvZME antibody,followed by HRP-GAR, then substrate2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) solutionwith 1 μg/ml 30% H₂O₂ was added. Absorbance at 405 nm was measured after30 min.

Example 9 Mehthods—Cytoxicity Assays in Vitro against Pae-Flt-1 andPae-FLK-1 for Granzyme B-vegf121 and against A375-M for GranzymeB-Scfvmel

[0364] PAE cells in Ham's F-12 medium with 10% FBS or A375-M cells inMEM medium with 10% FBS were plated into 96-well plates at a density of2.5×10³ cells per well and allowed to adhere for 24 hr at 37° C. in 5%CO₂. After 24 hr, the medium was replaced with medium containingdifferent concentrations of granzymeB-vegf121 or granzyme B-scFvMEL.After 72 hr, the effect of granzymeB-vegf121 or granzymeB-scFvMEL on thegrowth of cells in culture was determined using crystal violet staining.Surviving adherent cells were stained by adding 100 μl of crystal violet(0.5% (w:v) in ethanol). The stain was incubated on the plates for 0.5hr, excess stain was removed, and the plates were washed with water andallowed to air-dry. The remaining dye was solubilized by addition of 150μl of Somson's buffer (0.1 M sodium citrate, pH4.2). Plates were read ona microplate ELISA reader at 630 nM.

Example 10 In Vitro Transcription and Translation and in Vitro Cleavageof Procaspase 3 or DFF45 by Granzyme B or Bax Fusion Protein

[0365] An expression plasmid containing cDNA encoding procaspase-3 orDFF45 will be linearized with a restriction endonuclease digested and³⁵S-labeled procaspase-3 or DFF45 protein will be generated using an invitro rabbit reticulocyte TNT kit (Promega) according to themanufacturer's instructions. In brief, the linear plasmid containingcDNA encoding procaspase-3 or DFF45 (1 μg) will be incubated with theTNT reaction mixture containing 25 μl of rabbit reticulocyte lysate, 2μl of TNT reaction buffer, 1 μl of T7 RNA polymerase, 1 mM amino acidmixture minus cysteine, 2 μl of [³⁵S] cysteine or 2 μl of [³⁵S]methionine (10 mCi/ml) and 40 U of RNase inhibitor (Amersham PharmaciaBiotech, Inc.) in a total volume of 50 μl for 90 min at 30° C. For invitro cleavage, the translation products will be incubated in thepresence of granzyme B fusion or Bax fusion proteins in 150 mM NaCl. Thereactions will be performed at 30° C. in a final volume of 10 μl forvarious time intervals. The reactions will be then stopped by theaddition of an equal volume of 2×Laemmli buffer. Cleavage products willbe then separated by 15% SDS-PAGE and detected either by immunoblottingor phosphorimaging of the dried gels.

Example 11 Apoptosis Assays

[0366] Cell Morphology

[0367] A375-M or AAB527 cells (for Granzyme B-scFvMEL) and PAE-Flk1 orPAE-Flt1 cells (for Granzyme B-vegf121) will be grown in appropriatecell culture media. Cell death will be monitored by XTT assay. Tovisually monitor granzyme B-mediated or Bax-mediated apoptosis of thesecells, 1×10⁴ cells will be plated in each well of a 12-well microscopeslide. Forty-eight hours later, cells will be washed once in PBS, thentreated with 25 μl of serum-free medium supplemented with 1 μg/ml DTTand granzyme B fusion proteins or Bax fusion proteins. Followingincubation at 37° C. for 1 hr, supernatants will be removed and replacedwith 50 μl of complete medium. After a further 2 h at 37° C., cells willbe gently washed with PBS and fixed with acetone: methanol (1:1) for 2min at room temperature. Apoptosis of adherent cells will be visualizedby phase-contrast microscopy.

[0368] Assay of DNA Fragmentation

[0369] To monitor DNA fragmentation, 5×10⁵ cells in 50 μl of serum-freemedium will be supplemented with 1 μg/ml DTT and granzyme B fusionproteins or Bax fusion proteins. Following incubation at 37° C. for 1hr, they will be washed once in PBS. Fragmented DNA will be extractedusing a phenol/chloroform extraction assay. Briefly, the cell pelletwill be re-suspended in 25 μl of PBS, and an equal volume ofpheno/chloroform/isoamylalcohol (1:1:0.1) added. Following gentleagitation and centrifugation (10,000 g for 2 min), fragmented DNA willbe recovered, treated with RNase A for 1 hr at 37° C. and analyzed on 2%agarose gels containing ethidium bromide.

[0370] Facs Analysis

[0371] Cells (5×10⁵/10 ml) will be centrifuged at 450×g for 6 min,washed with cold PBS and resuspended in 300 μl of PBS. The cells will befixed with 5 ml methanol and left at −20° C. for at least 1 hr. Thecells will be then centrifuged at 800×g for 5 min., resuspended in 100μl PBS and diluted to a final volume of 1 ml with PBS. Cells will beincubated on ice for an additional 30 min, centrifuged at 800×g for 5min and resuspended in 0.5 ml PBS. 10 μl RNase (50 μg/ml) and propidiumiodide (PI, 5 μg/ml) will be added to the cell samples which will bethen FACS analyzed for DNA content as a function of cell number.

[0372] Cleavage of Caspase-3, PARP and DFF Detected in Cell SamplesTreated with Fusion Proteins vs. Non-treated with Fusion Proteins withinDifferent Time Course or Dose by using Western Blot Analysis

[0373] For western blotting, samples will be separated byelectrophoresis using 14% SDS-PAGE. The proteins will be transferredfrom gels into nitrocellulose membranes. The membranes will be blockedwith 5% non-fat milk, and incubated for 1 hr at room temperature withanti-caspase-3 or cleaved caspase-3, or anti-PARP or anti-cleaved DFFantibody (obtained from Cell Signaling Technology). After washing, themembranes will be incubated with goat anti-rabbit or goatanti-mouse/horseradish peroxidase conjugate. After further washing, themembranes will be developed using the Amersham ECL detection system andexposed to X-ray film.

Example 12 Signal Transduction Pathway-Non-Apoptosis Assays

[0374] In the following example, experiments are described which areperformed to analyze insulin signal transduction pathways, which arenon-apoptotic pathways, in cells treated with a delivery vehiclecontaining protein kinase B, which is a critical point in the insulinsignal transduction cascade leading to modulation of the enzyme glycogensynthetase kinase-3 (GSK-3). In the present invention, a fusionconstruct of the cytokine human hepatocyte growth factor(HCF) and thesignal transduction regulator protein kinase B(PKB) will be generated.The HCF component serves to bind to hepatocytes and to deliver activePKB to the cellular cytoplasm. From there, activation of the downstreammodulators of insulin signaling (GSK-3) will be assessed as describedbelow.

[0375] Anti-GSK3 antibodies are obtained from Transduction Laboratory.Phosphotyrosine antibody 4G10 and anti-PKB antibody will be obtainedfrom Upstate Biotechnology. Phosphospecific antibody against Ser-9 ofGSK3 will be obtained from Quality Controlled Biochemicals.

[0376] Cultures of hepatocytes in media will be treated with variousdoses of HCF or the HCF/PKB fusion construct. At various times afterdrug addition, cells will be harvested. To prepare cytosolic fractions,cells will be washed and collected in ice-cold phosphate-bufferedsaline. Cell pellets will be resuspended in ice-cold hypotonic buffer(25 mM Tris, pH 7.5, 1 mM EDTA, 25 mM NaF, 1 mM dithiothreitol) withComplete protease inhibitor mixture (Roche Molecular Biochemicals;Indianapolis, Ind.). Cells will be lysed after incubating on ice for 10min (verified by microscope analysis). The lysates will be subjected toultracentrifugation at 100,000×g for 30 minutes at 4° C., and thesupernatant will be collected. For immunoprecipitation, cells will bewashed twice in ice-cold phosphate-buffered saline, and then lysed in IPbuffer (125 mM NaCl, 25 mM NaF, 25 mM Tris, pH 7.5, 1 mM EDTA, 1 mMEGTA, 1% Triton X-100, 10 mM -glycerol phosphate, 5 mM sodiumpyrophosphate, 1 mM NaVO3, 200 nM okadaic acid, 1 mM dithiothreitol)with Complete protease inhibitor mixture. Anti-GSK3 antibody will beadded to clarified lysates for 1 h at 4° C., and then Protein G beads(Sigma) will be added for another 1 h. Immunoprecipitates will be washedthree times with IP buffer.

[0377] For GSK3 kinase assays, cells treated with the delivery vehiclecontaining active protein kinase B fusion construct GSK3immunoprecipitates will be washed once with kinase buffer (25 mM Tris,pH 7.5, 10 mM MgC12) first. Kinase reactions will be performed in kinasebuffer with 100 μM [γ-³² P]ATP and 100 μM 2BSP peptide as the substrate(synthesized by the Biomedical Resource Center, University ofCalifornia, San Francisco, Calif.). 2BSP is based on the GSK3 targetsite in eIF2B. After 20 min at 30° C., the reactions will be spotted onphosphocellulose P81 paper (Whatman), washed four times with 100 mMphosphoric acid, and counted in scintillation counter.

Example 13 Human Granzyme B Gene Cloning from Hut78

[0378] Native human granzyme B is a cytotoxic lymphocyte granule serineproteinase produced by cytotoxic T cells and natural killer cells.Initial attempts to clone human granzyme B gene from HL-60 cells, whichare promyelocytic leukemia cells from human peripheral blood, wereunsuccessful. However, the targeted cDNA, as a pre-mature granzyme Bgene, was obtained from human cutaneous T cell lymphoma Hut78 cells, byisolating RNA and using RT-PCR. A 1% agarose gel electrophoresis showedthat human pre-mature granzyme B cDNA was ˜800 bp (FIG. 1). The genesequence and amino acid sequence (FIG. 2) showed that the first 20 aminoacids are signal sequence. The human granzyme B sequence with signalsequence is designated pre-mature granzyme B. In cytotoxic cells, activegranzyme B is generated from a zymogen by dipeptidyl peptidase I(DPPI)-mediated proteolysis (Smyth et al., 1995). This removes thetwo-residue (Gly¹⁹Glu²⁰) propeptide and exposes Ile²¹ to become themature, N-terminal Ile-Ile-Gly-Gly sequence granzyme B.

Example 14 Construction of Granzyme B-Vegf121 or Granzyme B-Scfvmel

[0379] PCR was used to amplify the coding sequence of granzyme B fromIle²¹, which is the first residue of the mature enzyme, effectivelydeleting the signal sequence and GlyGlu domain. Concomitantly, acleavage site was inserted for enterokinase (AspAspAspLys; SEQ ID NO:53)upstream and adjacent to Ile^(21.) Granzymne B was attached to therecombinant Vegf121 or scFvMEL via flexible tether (G4S). The fused genefragment was then introduced into the Xba I and Xho I sites of thepET32a (+) to form the expression vector pET32GrB-vegf121 (FIG. 3A) andpET32GrB-scFvMEL (FIG. 3B). This vector contains a T7 promoter forhigh-level expression followed by a Trx.tag, a His.tag, a thrombincleavage site, and an enterokinase cleavage site for final removal ofthe protein purification tag (FIG. 4A and 4B). Once the protein tag isremoved by recombinant enterokinase, the first residue Ile of maturegranzyme was exposed, and the granzyme B moiety of granzyme B-vegf121 orgranzyme B-scFvMEL was activated. The nucleotide sequences and aminoacid sequences of granzyme B-vegf121(1059 base pairs, 353 aa) (FIGS. 4Cand 4D) and granzyme B-scFvMEL (1440 base pairs, 480 aa) (FIGS. 4E and4F) were confirmed.

Example 15 Expression and Purification of Granzyme B-VEGF121 or GranzymeB-SCFVMEL Fusion Protein

[0380] The recombinant protein granzyme B-vegf121or granzyme B-scFvMELwas expressed as polyhistidine-tagged protein designedpro-granzymeB-vegf121 or pro-granzyme B-scFvMEL and then purified byNickel-NTA metal affinity chromatography. The his-tag was cleaved byaddition of rEK to form granzymeB-vegf121 or granzyme B-scFvMEL. Oneliter of the culture typically yielded approximately 100 μg of the finalpurified granzymeB-vegf121 product and 150 μg of the final purifiedgranzyme B-scFvMEL product.

[0381] The results showed induced expression of granzyme B-relatedfusion constructs. The induced band at ˜55 kDa for granzyme B-vegf121and at ˜72 kDa for Granzyme B-scFvMEL represent, respectively, thegranzyme B-vegf121 or granzyme B-scFvMEL construct containing a ˜18 kDapurification tag. Enzymatic digestion of the tag using recombinantenterokinase (rEK) resulted in appearance of a band migrating at ˜38 kDafor granzyme B-vegf121 and at ˜53 kDa for granzyme B-scFvMELrepresenting native proteins. Thus, SDS-PAGE showed that the finalpurified granzymeB-vegf121 fusion construct migrated under reducingconditions as a band at the expected molecular weight of 38 kDa (FIG.5A) and granzyme B-scFvMEL fusion construct showed the band at theexpected molecular weight of 53 kDa (FIG. 5B).

[0382] Specificity of the cleaved fusion protein was confirmed byWestern blot using either mouse anti-granzyme B monoclonal antibody,mouse anti-vegf121 polyclonal antibody, or rabbit anti-scFvZMEpolyclonal antibody (FIG. 6). The results showed that granzyme B-vegf121fusion construct (FIG. 6A) could specifically bind to either mouseanti-vegf121 antibody (FIG. 6A (a)) or mouse anti-granzyme B monoclonalantibody (FIG. 6A (b)). The molecular weights of pro-granzyme B-vegf121and Granzyme B-vegf121 are approximately 55 kDa and 38 kDa,respectively. Granzyme B-scFvMEL fusion construct (FIG. 6B) couldspecifically bind to either mouse anti-granzyme B monoclonal antibody(FIG. 6B (c)) or rabbit anti-scFvMEL polyclonal antibody (FIG. 6B (d)).The molecular weights of Pro-granzyme B-scFvMEL and Granzyme B-scFvMELare approximately 70 kDa and 53 kDa, respectively.

Example 16 Bindging Activity of Scfvmel Moiety of Granzyme B-SCFVMELFusion Protein

[0383] The scFvMEL moiety was tested to bind Protein L, which is animmunoglobulin-binding protein that originally comes from the bacteriaPeptostreptococcus magnus. Protein L has the unique ability to bindthrough kappa light chain interactions without interfering with anantibody's antigen-binding site. This gives Protein L the unique abilityto bind Single Chain Variable Fragments (scFv). The results showed theabsorbance at 405 nm concentration-response increase, suggesting scFvMELbound to Protein L. (FIG. 7). The binding activity of the granzymeB-scFvMEL was the same as that of the scFvMEL-TNF, which couldspecifically bind to antigen-positive human melanoma cells and wascytotoxic activity to those melanoma cells.

Example 17 In Vitro Cytotoxic Effects of Granzyme B-Vegf121

[0384] The cytotoxicity of GranzymeB-vegf121 was assessed againstlog-phase PAE-Flk-1 (Overexpression flk-1/KDR receptor) and PAE-Flt-1(overexpression flt-1 receptor) in culture, wherein 2.5×10³ cells perwell on 96-well plates. A 50% growth inhibitory effect was found at aconcentration of ˜10 nM on PAE-Flk-1 cells. However, no cytotoxiceffects were found on PAE-Flt-1 cells (FIG. 8). It was also shown thatVEGF121 could specifically bind to VEGF receptor Flk-1/KDR but not toFlt-1. The cytotoxicity of granzymeB-vegf121 demonstrated that theconstruct could specifically kill PAE-Flk-1 cells, which indicated thatthe Vegf121 moiety of the fusion bound to the Flk-1 over-expressioncell-surface. Subsequently, there was delivery of granzyme B to theinterior of targeted cells, resulting in cytotoxicity to the targetcells.

Example 18 In Vitro Cytotoxic Effects of Granzyme B-Scfvmel

[0385] The cytotoxicity of granzyme B-scFvMEL was tested againstlog-phase human melanoma A375-M cells. The results showed that granzymeB-scFvMEL could kill the A375-M cells, with an IC₅₀ concentration ofapproximately 20 nM. When pre-treated with scFvMEL-3825 at theconcentration of 178.5 nM for 6hr, followed by treatment with granzymeB-scFvMEL for 72 hr, a 15-fold higher concentration of granzymeB-scFvMEL was required to exhibit 50% cytotoxicity compared to theabsence of scFvMEL-3825 pre-treatment (FIG. 9). In a specificembodiment, this is because the cell-surface antigen gp240 was alreadyoccupied by scFvMEL-3825, resulting in a reduced chance for the scFvMELmoiety of granzyme B-scFvMEL binding to the gp240 antigen, consequentlyinhibiting the cytotoxicity of granzyme B-scFvMEL on these cells. Theresults suggested that the cytotoxicity, at least in part, is due to theinteraction of the antibody with its cell-surface domain.

Example 19 Cloning Human Bax Gene

[0386] Total RNA from Namalwa cells was isolated using Glass MAX RNAMicroisolation Spin Cartridge System (Gibco BRL). Removal of the genomicDNA was performed by addition of RNase-free DNase I while incubating atroom temperature for 15 min. DNase I then was inactivated by adding EDTAsolution and heating for 15 min at 65° C. SUPERSCRIPT First-StrandSynthesis System was utilized for RT-PCR (Gibco BRL). First-strandsynthesis used Oligo (dT), and then the target cDNA (Bax cDNA) wasamplified using the primers: NbaxTA (5′ to 3′): GGTGATGGACGGGTCCGGGGAGCA(SEQ ID NO:29) and CbaxTA (5′ to 3′): GGCCTCAGCCCATCTTCTTCCAGATGGTGA(SEQ ID NO:30) by PCR with the following cycles: denaturation at 95° C.for 5 min, 30 cycles of 94° C. for 1 min, 50° C. for 1 min, 72° C. for 1min, and extension at 72° C. for 5 min. A 1% agaro run to check the PCRproduct. Purified PCR fragment was cloned into PCR 2.1 TA vector(Invitrogen) and designed BaxTA. The BaxTA was transformed into INVαF′competent cells, and the positive clones were screened by blue/whitecolonies screening or by PCR methods. The DNAs for positive clones wereisolated by using QIApre Spin prep kit (Qiagen; Valencia, Cailf.), andsequencing confirmed the human bax gene in the correct clone (BaxTA-35(clone # 35)).

Example 20 Construction of Bax-related Fusion Genes

[0387] The construction was based on an over-lap PCR method. The scFvMELgenes were fused to Bax, truncated Bax1-5 (that is, comprises exons 1through 5) or truncated Bax345 (that is, comprises exons 3, 4, and 5)genes with G4S tether in different orientation (designated scFvMEL-baxor Bax-scFvMEL, scFvMEL-Bax1-5 or Bax1-5-scFvMEL, and scFvMEL-Bax345 orBax345-scFvMEL, respectively). As shown in FIG. 10, a skilled artisanrecognizes that the human Bax gene (SEQ ID NO:45), which encodes thepolypeptide of SEQ ID NO:46, comprises six exons, with the domain BH1(DGNFNWGRVVA; SEQ ID NO:47) in exon 4, BH2 (WIQDQGGWD; SEQ ID NO:48) inexon 5, and BH3 (LKRIGDE; SEQ ID NO:49) in exon 3. In a specificembodiment, the Bax chimeric polypeptide comprises the BH1, BH2 and BH3domains or a combination thereof. In another specific embodiment, theBax chimeric polypeptide consists essentially of the BH1, BH2 and BH3domains. In another specific embodiment, the Bax chimeric polypeptidecomprises exons 3, 4, and 5 or a combination thereof. In an additionalspecific embodiment, the Bax chimeric polypeptide consists essentiallyof exons 3, 4, and 5.

[0388] Briefly, scFvMEL coding sequence was amplified frompET32a-scFvMEL/TNF by PCR and full length bax or truncated Bax1-5 ortruncated Bax345 was amplified from BaxTA-35 by PCR. Different primerswere designed wherein G4S liner sequence was added to the C-terminus orN-terminus in order to link the fused genes together by the second PCR.In order to clone the fused genes into pET32a (+) vector at Nco I andXho I sites, primers added the Nco I site at the N-terminus and two stopcodons, and a Xho I site was added at the C-terminus. The first PCR wereperformed by 95° C. for 5 min, 30 cycles of 94° C. for 1 min, 50° C. for1 min and 72° C. for 1 min, and then extension at 72° C. for 5 min. Forconstructing of scFvMEL-bax, scFvMEL was amplified by using primers:Ncolzme (5′ to 3′): GGTGGCGGTGGCTCCATGGCGGACATTGTGATGACCCAGTCTCAAAAATTC(SEQ ID NO:31) and Czme (5′ to 3′):CGTCGGAGCCACCGCCACCGCTAGCTGAGGAGACGGTGAGAGT (SEQ ID NO:32), Bax fragmentwas amplified by using primers: Nbax2 (5′ to 3′):GGTGGCGGTGGCTCCGACGGGTCCGGGGAGCAG (SEQ ID NO:33) and Cbax (5′ to 3′):GGAGCCACCGCCACCCTCGAGCTATCAGCCCATCTTCTTCCAGAT (SEQ ID NO:34). ForBax-scFvMEL construct, primers are Nbax (5′ to 3′):GGTGGCGGTGGCTCCATGGACGGGTCCGGGGAGCAG (SEQ ID NO:35), Cbax2-1 (5′ to 3′):GTCCGTGGAGCCACCGCCACCGCTAGCGCCCATCTTCTTCCA (SEQ ID NO:36), Nzme2 (5′ to3′): GGTGGCGGTGGCTCCACGGACATTGTGATGACCCAGTCTCAAAAATTC (SEQ ID NO:37) andCzme2 (5′ to 3′): GGAGCCACCGCCACCCTCGAGCTATCATGAGGAGACGGTGAGAGTGGT (SEQID NO:38). For construction of scFvMEL-Baxl-5 construct, primers areNcolzme, Czme, Nbax2 and Cxholbax345 (5′ to 3′):GGAGCCACCGCCACCCTCGAGCTATCACCAACCACCCTGGTC (SEQ ID NO:39). Forconstruction of Bax1-5-scFvMEL fusion construct, primers are Nbax,Cbax345 (5′ to 3′): GGAGCCACCGCCACCCCAACCACCCTGGTC (SEQ ID NO:40), Nzme2and Czme2. For construction of scFvMEL-Bax345fusion, primers areNcoIzme, Primer3 (5′ to 3′): CCGGAGCCACCGCCACCGCTAGCTGAGGAGACTGTGA (SEQID NO:41), Nbax345 (5′ to 3′): GGTGGCGGTGGCTCCTTCATCCAGGATCGAG (SEQ IDNO:42), and CxhoIbax345. For construction of Bax345-scFvMEL, NcoIBax345(5′ to 3′) is used: GGTGGCGGTGGCTCCATGGTCATCCAGGATCGAG (SEQ ID NO:43),Cbax345, Nzme2 and Czme2.

[0389] Then, the second PCR was performed by 30 cycles of 94° C. for 1min, 50° C. for 1 min and 72° C. for 1 min, further extension at 72° C.for 5 min. Amplified fragments were separated by 1% agarose gelelectrophoresis, purified by PCR purification kit (Qiagen; Valencia,Cailf.). The purified PCR products were digested with Nco I and Xho I at37° C. for 3 hrs and then separated by 1% agarose gel electrophoresis,purified from the gels using geneclean II kit (Quantium Biotechnologies,Inc., Carlsbad, Calif.). The purified gene fragments were cloned intopET32a (+) vector, the ligation mixture was transformed into DH5αcompetent cells, screened the positive clones by PCR, then sequenced.The clones with correct-frame sequence were transformed into AD494(DE3)pLysS competent cells for further induction and expression.

[0390] For expression of full length Bax and Bax-scFvMEL protein, theBax and Bax-scFvMEL sequences were subdloned into pBAD/His A vector anddesignated pBAD/Hisbax and pBAD/Hisbax-scFvMEL, respectively. Briefly,the full length Bax and Bax-scFvMEL genes were amplified by using a PCRmethod. For amplification of full length Bax, BaxTA-35 was used astemplate, and the primers were NBAXHIS (5′ to 3′):AAACATGCCATGGCTCACCACCACCACCACCACGACGGGTCCGGGGAGCAGCCC AGA (SEQ IDNO:44) and Cbax. For amplification of Bax-scFvMEL, pET32-Bax-scFvMEL(clone2) was used as template, and primers were NBAXHIS and Czme2. TheNBAXHIS primer was designed as follows: Nco I site for cloning,polyhistidine (6×His) for purification and detection at the N-terminus,followed by the initiation ATG; two stop codons and a Xho I site wereadded at the C-terminus in Cbax and Czme2 primers. Purified PCRfragments were digested by Nco I and Xho I and purified by using geneclean kit, following ligation into the same restriction endonucleasesdigested for the pBAD/His A vector. The ligation mixture was transformedinto DH5α, and the positive clones were screened by PCR screening, DNAwas isolated, and the sequence was checked. The confirmed sequencepositive clones were transformed into LMG194 competent cells forexpression.

Example 21 Induction and Expression of Bax-related Fusion Proteins In E.Coli

[0391] Bacterial colonies transformed with the constructed plasmid weregrown in Luria Broth (LB) growth media containing 200 μg/ml ampicillin,70 μg/ml chloramphenicol, and 15 μg/ml kanamycin, at 37° C. overnight at24 rpm shaking. The cultures were then diluted 1:100 in fresh LB mediaplus antibiotics and grown to A₆₀₀=0.6 at 37° C., thereafter, induced byaddition of IPTG to a final concentration of 80 μM at 37° C. for 2 hrs.The cells were harvested, resuspended in 10 mM Tris (pH 8.0) and storedfrozen at −80° C. for further purification.

Example 22 Induction and Expression of Full Length Bax and Bax-scfvmel

[0392] Bacterial colonies transformed with the plasmid pBAD/Hisbax andpBAD/Hisbax-scFvMEL were grown in RM medium containing glucose and 100μg/ml ampicillin, at 37° C. overnight with shaking (225 rpm). Thecultures were then diluted by 1: 100 in fresh RM medium containingglucose and 100 μg/ml ampicillin and grown at 37° C. with shaking tomaxium OD₆₀₀, then induced by addition of 5% arabinose at 37° C. for 4hours. The cells were harvested, resuspended in 10 mM Tris (pH 8.0) andstored frozen at −80° C. for further purification.

Example 23 Purification of Bax-related Fusion Proteins

[0393] Re-suspension was lysed by addition of lysozyme to a finalconcentration of 100 μg/ml tumbling for 30 min at 4° C., followed bysonication. Extracts were centrifuged at 10,800 g for 30 min, and thesupernatant was further centrifuged at 40,000 rpm for 1 hr. Thesupernatant containing only soluble protein was adjusted to 40 mM Tris,pH 8.0, 10 mM Imidazole and was applied to a Nickel-NTA agaroseequilibrated with the same buffer. After washing the Nickel-NTA agarosewith 500 mM NaCl 20 mM imidazole, the bound proteins were eluted with500 mM NaCl 500 mM imidazole. Absorbance (280nm) and SDS-PAGE analysiswere performed to determine the polyhistidine-tagged proteins. The finalproteins were obtained by the addition of recombinant bovineenterokinase (rEK) to remove the polyhistidine-tag according to themanufacturer's instruction (1 unit of rEK cleavage 50 μg protein,incubated at room temperature for 16 hrs). The rEK was then removed byEK capture agarose. The final proteins were analyzed by SDS-PAGE andstored at 4° C.

Example 24 SDS-page and Western Blot Analysis

[0394] Protein samples were analyzed by electrophoresis on a 8.5% or 12%SDS-PAGE under reducing conditions. The gels were stained with CoomassieBlue. For western blotting analysis, proteins were transferred from gelsinto nitrocellulose membranes. The membranes were blocked with 5%non-fat milk and incubated for 1 hr at room temperature or overnight at4° C. with rabbit anti-scFvzme antibody (1:2000 dilution) or rabbitanti-bax antibody (1:1000 dilution). After washing, the membranes wereincubated with goat anti-rabbit/horseadish peroxidase conjugate(HRP-GAR, 1:5000 dilution). After further washing, the membrane wasdeveloped using the Amersham ECL detection system and exposed to X-rayfilm.

Example 25 Detection of Scfvmel Moiety of Bax-related Fusion Proteins

[0395] Reacti-Bind TM Protein L Coated Plates from PIERCE (Rockford,Ill.) or 96-well plates containing adherent human melanoma A375-M cellswere used for detection of scFvMEL moiety of Bax-related fusionproteins, based on ELISA method. Briefly, Pre-coated Protein L wasblocked by 5% BSA and then reacted with scFvMEL bax-related fusionproteins at various concentration. After washing, they were incubatedwith rabbit anti-scFvZME antibody, followed by incubation withHRP-GAR(1:5000 dilution) for 1 hr at room temperature, and thensubstrate 2,2′ -azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)solution with 1 μg/ml 30% H₂O₂ added. Absorbance at 405 nm was measuredafter 30 min.

Example 26 In Vitro Cytotoxicity Assays

[0396] Human melanoma A375-M cells cultured in MEM medium with 10% FBSwere plated into 96-well plates at a density of 2.5×10³ cells per welland allowed to adhere for 24 hr at 37° C. in 5% CO₂. After 24 hr, themedium was replaced with medium containing different concentrations ofdifferent scFvMEL-bax-related fusion proteins. After 72 hr, the effectof those fusion proteins on the growth of cells in culture wasdetennined using crystal violet staining, or XTT. Plates were read on amicroplate ELISA reader at 540 nm.

Example 27 Cytotoxicity of Bax Chimeric Polypeptides

[0397] As demonstrated in FIG. 11, the human bax gene was cloned by PCRfrom Namalwa cells. A 1% agarose gel electrophoresis demonstrating humanBax cDNA synthesized from Namalwa cells by RT-PCR.

[0398]FIGS. 12A and 12B illustrate construction of scFvMEL Bax-relatedfusion proteins. The Bax gene consists of six exons, and the geneproduces alternative transcripts, the predominant form of which encodesa 1.0 kb mRNA and transcript 21 kDa protein which designated Bax α. Theboxes indicate exons identified by numbers. Exon 6 is the transmembranedomain. The scFvMEL genes were fused to Bax, truncated Bax1-5 or Bax345with G4S tether in two different orientations, designated scFvMEL-bax,Bax-scFvMEL, scFvMEL-bax1-5, Bax1-5-scFvMEL, scFvMEL-bax345 andBax345-scFvMEL. The fusion genes were cloned into pET32a(+) expressionvector at Nco I and Xho I sites. Then the plasmid containing fusiongenes was transformed into AD494(DE3)pLysS E. coli for expression.

[0399]FIG. 13 demonstrates western blot analysis illustrating expressionof scFvMEL Bax-related fusion proteins in pET32a (+) vector. TheSDS-PAGE Coomassie Blue staining of truncated bax-related proteinsoccurred under reducing conditions. The results showed inducedexpression of scFvMEL and truncated bax fusion proteins in pET32a(+)expression vector. The induced bands were at ˜62 kDa for scFvMEL-bax345and Bax345-scFvMEL and were at ˜65 kDa for scFvMEL-Bax1-5 andBax1-5-scFvMEL, respectively, which also contained a ˜20 kDapurification tag.

[0400]FIG. 14 shows the expression of pET32-scFvMEL-bax andpET32-Bax-scFvMEL transformed into AD494(DE3)pLysS E coli and under IPTGinduction. The full-length bax fusion proteins are very toxic to thebacteria because of the highly hydrophobic domain-exon 6. The bacteriacontaining the plasmid pET32-scFvMEL-Bax or pET32-Bax-scFv were grown inLB media containing 200 μg/ml ampicillin, 70 μg/ml chloramphenicol and15 μg/ml kanamycin to OD₆₀₀=0.6, induced by addition of IPTG to thefinal concentration of 0.1 mM, the bacteria died in terms of thedecrease value of OD₆₀₀.

[0401]FIG. 15 demonstrates the expression of the full length bax andBax-scFvMEL in pBAD/HisA vector transformed LMG194 E coli. The fulllength bax gene and Bax-scFvMEL gene were cloned into pBAD/His A vectorat Nco I and Xho I sites. The polyhistidine (6 his) tags were followedby the initiation ATG. The plasmids containing either Bax or Bax-scFvMELwere transformed into LMG194 cells for expression, and the expression ofthe Bax and Bax-scFvMEL proteins was tested in different bacterialgrowth media (RM containing glucose and 100 μg/ml ampicillin, RMcontaining 100 μg/ml ampicillin, and LB containing 100 μg/mlampicillin). The LMG194 transformed pBAD/HisLacZ was used as a positivecontrol, with an expression band at ˜120 kDa representing LacZ proteinwhich could be detected by anti-bax antibody. The LMG194 transformedpBAD/HisA (empty vector) was used as a negative control. Westernblotting was performed using rabbit anti-Bax monoclonal antibody (1:1000dilution) as the primary antibody and HRP-Goat-anti-rabbit IgG as thesecondary antibody. The results showed that the bands at 21 kDarepresent Bax with 6X His-tag, and the bands at ˜49 kDa representBax-scFvMEL with 6X His-tag.

[0402] Binding activity of scFvMEL moiety of the fusion proteins isdemonstrated in FIGS. 16A and 16B. A375-M cells are gp240antigen-positive human melanoma cell lines. Protein L has the uniqueability to bind scFv. The fusion protein Bax345-scFvMEL could bind toeither A375-M (FIG. 16A) or Protein L (FIG. 16B) detected withanti-scFvzme antibody. This binding activity is the same as the otherfusion protein scFvMEL-TNF.

[0403]FIG. 17 demonstrates cytotoxicity of scFvMEL-bax345 vs.Bax345-scFvMEL on A375-M. The cytotoxic effects of scFvMEL-bax345 andBax345-scFvMEL against log-phase human antigen-positive melanoma A375-Mcells were assessed. A375-M cells were set up in 96-well plates (2.5×10³cells per well). The IC₅₀ concentrations of scFvMEL-bax345 andBax345-scFvMEL were approximately 3 nM and 10 nM, respectively.

Example 28 Elisa of Granzyme B-Vegf121 on Various Cell Lines (Detectedwith Mouse Anti-vegf121 Antibody)

[0404] ELISA Assay of Binding Activity utilized 96-well platescontaining adherent PAE/flk-1, PAE/flt-1, A375-M or SKBR3-HP cells thatwere blocked by 5% BSA and then reacted with purified Granzyme B-vegf121at various concentrations. After washing, they were incubated with mouseanti-vegf antibody, followed by HRP-labeled goat anti-mouse IgG. Then asa substrate 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)solution with 1 μl/ml of 30% H₂O₂ was added. Absorbance at 405 nm wasmeasured after 30 min.

[0405] VEGF121 can specifically bind to the flk-1/KDR receptor on thevascular endothelial cells (FIG. 18). The experiment showed that thefusion protein Granzyme B-vegf121 specifically binds to PAE/fik-1 cellswhich overexpress flk-1 receptors, but there were no binding activitieson PAE/flt-1 (overexpressed flt-1 receptors) or human melanoma A375-Mcells and human breast cancer SKBR3-HP cells. That is, GrB/VEGF121 canspecifically bind to PAE/flk-1 cells which overexpress flk-1/KDRreceptor detected with either VEGF121 antibody or GrB antibody

Example 29 Cytotoxicity of Granzyme B/vegf121 on Transfected EndothelialCells

[0406] Cytotoxicity of Granzyme B-VEGF121 against PAE/flk-1 vs PAE/flt-1cells was assessed against log-phase PAE/flk-1 (transfected withflk-1/KDR receptor) and PAE/flt-1 (transfected with flt-1 receptor) inculture. Briefly, PAE cells were plated into 96-well plates at a densityof 2.5×10³ cells per well. After 24 hr, the cells were treated withmedium containing different concentrations of Granzyme B-VEGF121. Theeffect on the growth of cells was determined using XTT after 72 hrs.Plates were read on a microplate ELISA reader at 546 nm.

[0407] The results showed that a 50% growth inhibitory effect (I.C.₅₀)was found at a concentration of 10 nM on PAE/flk-1 cells (FIG. 19).However, no cytotoxic effects were found on PAE/flt-1 cells. Thecytotoxicity of Granzyme B-VEGF121 on PAE/flk-1 cells indicates that theVEGF121 moiety of the fusion specifically bound to flk-1 over-expressionon the cell-surface, followed by delivery of granzyme B to the interiorof targeted cells and cytotoxicity to the target cells.

Example 30 Cytotoxicity Assay of Granzyme B-VEGF121 vs VEGF121-rGel inVitro Against PAE/FLK-1

[0408] PAE cells in Ham's F-12 medium with 10% FBS were plated into96-well plates at a density of 2.5×10³ cells per well and allowed toadhere for 24 hr at 37° C. in 5% CO₂. After 24 hr, the medium wasreplaced with medium containing different concentrations of GranzymeB-VEGF121 or VEGF121-rGel. After 72 hrs, the effect of GranzymeB-VEGF121 or VEGF121-rGel on the growth of cells in culture wasdetermined using XTT. Plates were read on a microplate ELISA reader at540 nm.

[0409] The results showed that the I.C.₅₀ of VEGF121-rGel wasapproximately 1 nM (FIG. 20). The I.C.₅₀ of Granzyme B-VEGF121 was10-fold higher than that of VEGF 121-rGel.

Example 31 Caspase Activity of Pae Cells Treated with Granzyme B-VEGF121

[0410] Western Blotting analysis of caspase activation was carried outwith 30 μg of whole cell lysates. Following SDS-PAGE, the proteins wereelectrophoretically transferred onto nitrocellulose membranes. Themembranes were blocked with phosphate-buffered saline with 0.5% Tween 20(PBST) containing 5% fat-free milk and then exposed to caspase-8,caspase-3, caspase-6, caspase-7, cleaved caspase-3, PARP or cleavedDFF45 antibodies, respectively. The membranes were washed with PBST andtreated with secondary antibodies conjugated to horseradish peroxidase.The antigen-antibody reaction was visualized by an enhancedchemiluminescence (ECL) assay using Amersham ECL develop reagents andexposure to film.

[0411] The results showed that after a 4 hr treatment by GranzymeB-VEGF121, cleaved caspase-8, cleaved caspase-3, cleaved PARP andcleaved DFF45 were observed on PAE/flk-1 cells but not on PAE-flt-1cells (FIG. 21). However, caspase-6 or caspase-7 was not cleaved byGranzyme B-VEGF121, which indicated that Granzyme B-VEGF121 activatedcaspases only involved in caspase-8, caspase-3 apoptosis pathway.

Example 32 In Situ Cell Death Detection (Tunel)

[0412] Cleavage of genomic DNA during apoptosis may yielddouble-stranded, low molecular weight DNA fragments as well as singlestrand breaks (nicks) in high molecular weight DNA. Those DNA strandbreaks can be identified by labeling free 3′-OH termini with modifiednucleotides in an enzymatic reaction. This method uses terminaldeoxynucleotidyl transferase (TdT), which catalyzes polymerization ofnucleotides to free 3′-OH DNA ends in a template-independent manner, isused to label DNA strand breaks. Incorporated fluorescein is detected byanti-fluofescein antibody Fab fragments from sheep, conjugated withalkaline phosphatase (AP). After substrate reaction, stained cells canbe analyzed under light microscope.

[0413] For the cell treatments, cells were plated onto 16-well chamberslides, 2500 cells/well, incubated for overnight at 37° C./5% CO₂conditions. Cells were treated with fusion protein GrB-scFvMEL orGrB-vegf121 at I.C-₅₀ concentration for different times (24, 48 hr,etc.) and washed briefly with PBS.

[0414] For the TUNEL Assay, cells were fixed with 3.7% formaldehyde atroom temperature for 20 min, after rinsing with PBS, permeabilzed with0.1% Triton X-100, 0.1% sodium citrate on ice for 2 min and then washedwith PBS twice. Cells were incubated with TUNEL reaction mixture at 37°C. for 60 min, followed by incubation with Converter-AP at 37° C. for 30min, and finally reacted with Fast Red substrate solution at roomtemperature for 10 min. After final wash step, the slides were mountedin mounting medium and analyzed under light microscope.

[0415] Positive controls were included in each experimental set up.Fixed and permeabilized cells were incubated with 1 mg/ml of DNase I for10 min at 37° C. to induce DNA strand breaks.

[0416] The effect of GrB-scFvMEL on A375-M and SKBR3-HP cells by in situcell death detection (TUNEL) was determined. TUNEL positive results wereobserved with respect to GrB-scFvMEL treated antigen-positive humanmelanoma A375-M cells at 48-hr but not with respect to GrB-scFvMELtreated antigen-negative human breast cancer SKBR-3-HP cells. Thisindicated that GrB-scFvMEL could specifically target antigen-positivemelanoma cells and induce cell apoptosis.

[0417] The effect of GrB-vegf121 on PAE/Flk-1 vs. PAE/Flt-1 cells byTUNEL Assay was determined. VEGFR2/KDR was over-expressed on PAE/Flk-1but not PAE/Flt-1 cell surface. VEGF121 specifically targetedVEGFR2/KDR, delivering GrB into PAE/Flk-1 cells. TUNEL Assay positiveresults were observed with respect to GrB-vegf121 treated PAE/Flk-1 at24 hr and 48 hr but not with respect to GrB-vegf121 treated PAE/Flt-1.GrB-vegf121 induced PAE/Flk-1 cell apoptosis.

Example 33 Internalization of GrB/VEGF121 into Pae Cells byImmunofluorescence Microscopy

[0418] This example is directed to an internalization analysis ofGrB/VEGF121 by immunofluorescence microscopy. Cells were treated asfollows. Cells were plated in 16-well chamber slides (Nunc), 1×10⁴ cellsper well, incubated for overnight at 37 ° C./5% CO₂ conditions. Cellswere treated with 200 nM of GrB/VEGF121 for 4 h and then washed brieflywith PBS. The cell surface was stripped by incubations for 10 min withglycine buffer (500 mM NaCl, 0.1 M glycine, pH 2.5), neutralized for 2min with 0.5 M Tris, pH 7.4, and washed briefly with PBS.

[0419] Immunofluorescent staining occurred as follows. Cells were fixedin 3.7% formaldehyde for 15 min at RT, followed by a brief rinse withPBS and then permeabilization for 10 min in PBS containing 0.2% TritonX-100. They were then washed three times with PBS. Samples wereincubated with 3% BSA for 1 h at RT to block nonspecific binding sites,then incubated with mouse anti-granzyme B antibody (1:100 dilution) atRT for 1 h, followed by washing three times with PBS. The samples wereincubated with fluorescein isothiocyanate (FITC)-coupled anti-mouse IgG(Sigma) (1:100 dilution) at RT for 1 h and then washed three times withPBS. The walls and gaskets were removed carefuilly. After air drying,the slide was mounted and analyzed under a fluorescence microscope.

[0420] The results showed that the GrB moiety of GrBNVEGF₁₂₁ wasdelivered into the cytosol of PAE/flk-1 but not into that of PAE/flt-1cells after 4 h treatment.

Example 34 GrB/VEGF121 Induces Apoptosis on Pae/Flk 1 Cells Detected byTunel Assay

[0421] Cleavage of genomic DNA during apoptosis may yielddouble-stranded, low molecular weight DNA fragments as well as singlestrand breaks (nicks) in high molecular weight DNA. Those DNA strandbreaks can be identified by labeling free 3′-OH termini with modifiednucleotides in an enzymatic reaction. This method uses terminaldeoxynucleotidyl transferase (TdT), which catalyzes polymerization ofnucleotides to free 3′-OH DNA ends in a template-independent manner, tolabel DNA strand breaks. Incorporated fluorescein is detected byanti-fluorescein antibody Fab fragments from sheep conjugated withalkaline phosphatase (AP). After substrate reaction, stained cells canbe analyzed under light microscope.

[0422] Cell treatments were as follows. Cells were plated onto 16-wellchamber slides, 2500 cells/well, incubated for overnight at 37° C./5%CO₂ conditions. Cells were treated with fusion protein GrB-vegf121 atI.C.₅₀ concentration for different times (24, 48 hr, etc) and washedbriefly with PBS.

[0423] The TUNEL Assay was as follows. Cells were fixed with 3.7%formnaldehyde at room temperature for 20 min, followed by rinsing withPBS and permeabilization with 0.1% Triton X-100, 0.1% sodium citrate onice for 2 min. They were then washed with PBS twice. Cells wereincubated with TUNEL reaction mixture at 37° C. for 60 min, followed byincubation with Converter-AP at 37° C. for 30 min, and finally reactedwith Fast Red substrate solution at room temperature for 10 min. Afterfinal wash step, the slides were mounted in mounting medium and analyzedunder light microscope.

[0424] The results indicated that VEGFR2/KDR over-expressed on PAE/Flk-1but not the PAE/Flt-1 cell surface. VEGF121 specifically targetedVEGFR2/KDR, delivering GrB into PAE/Flk-1 cells. TUNEL Assay positiveresults were demonstrated for GrB/VEGF1₂₁-treated PAE/Flk-1 at 24 hr and48 hr but not on GrB/VEGF121-treated PAE/Flt-1. Thus, GrB/VEGF121induced PAE/Flk-1 apoptosis.

Example 35 Cytochrome C Release of Pae Cells Treated with GrB/VEGF121

[0425] Cytochrome c plays an important role in apoptosis. The protein islocated in the space between the inner and outer mitochonial membranes.An apoptotic stimulus triggers the release of cytochrome c from themitochondria into the cytosol where it binds to Apaf-1. The cytochromec/Apaf-1 complex activates caspase-9, which then activates caspase-3 andother downstream caspases.

[0426] Materials and methods for the cytochrome c release apoptosisassay: (from Oncogene Research Products; San Diego, Calif.) was asfollows. PAE/flk-1 cells and PAE/flt-1 cells (5×10⁷) were treated withGrB/VEGF121 at concentrations of 0.1 nM and 20 nM for 24 h. Cells werecollected. After washing cells with 10 ml of ice-cold PBS, the cellswere resuspended with 0.5 ml of 1×cytosol extraction buffer mixcontaining DTT and Protease Inhibitors, and incubated on ice for 10 min.Cells were homogenized in an ice-cold glass homogenizer. The homogenatewas transferred to a 1.5 ml microcentrifuge tube and centrifuged at 700μg for 10 min at 4° C. The supernatant was transferred to a fresh 1.5 mltube and centrifuged at 10,000×g for 30 min at 4° C. Supernatant wascollected as a cytosolic fraction. The pellets were resuspended in 0.1ml mitochondrial extraction buffer mix containing DTT and proteaseinhibitors, vortexed for 10 seconds, and saved as a mitochondrialfraction. Protein concentrations were determined by using Bio-RadLaboratories, Inc. (Hercules, Calif.) Bradford Protein Assay. 10 μg ofeach cytosolic and mitochondrial fraction isolated from non-treated andtreated cells were loaded on a 15% SDS-PAGE, followed by standardWestern blot procedure and probing with cytochrome c antibody (1 μg ml).

[0427] For the cytochrome c release apoptosis assay, PAE cells weretreated with GrB/VEGF121 at different concentrations for 24 h. A highlyenriched mitochodrial fraction was isolated from the cytosol. Cytochromec translocation from mitochondria into cytosol during apoptosis wasdetermined by western blotting using cytochrome c antibody. FIG. 22shows cytochrome c release on PAE/flk-1 but not on PAE/flt-1 cells afterGrB/VEGF121 treatment.

Example 36 Bax Translocation of Pae Cells After GrB/VEGF121 TREATMENT

[0428] Bax, a 21 kDa protein with extensive amino acid homology withBcl-2, is variably expressed by different cells. Bax as a pro-apoptoticmember of the Bcl-2 family showed some structural similarities withpore-forming proteins. Hence, it is believed that Bax can formtransmembrane pores across the outer mitochondrial membrane, which leadsto a loss of membrane potential. The localization of Bax has been shownto change from the cytosol to the mitochondria upon the receipt of anapoptotic stimulus.

[0429] Isolation of the cytosolic fraction and mitochondrial fractionwas performed using an Oncogene Research Products kit (Cat # QIA87; SanDiego, Calif.). Protein concentrations were determined by using Bio-RadLaboratories, Inc. (Hercules, Calif.) Bradford Protein Assay. 10 μg ofeach cytosolic and mitochondrial fraction isolated from non-treated andtreated cells were loaded on a 12% SDS-PAGE, and then a standard Westernblot procedure was performed and probed with Bax antibody (Santa CruzBiotechnology, Inc.; Santa Cruz, Cailf.; 1:200 dilution).

[0430] Western blotting analysis was performed of Bax expression on PAEcells after GrB/VEGF121 treatment for 24 h. The results showed that thelocalization of Bax changed from the cytosol to the mitochondria onPAE/flk-1 but not on PAE/flt-1 cells after treatment with GrB/VEGF121 atthe concentration of 20 nM (I.C.₅₀). FIG. 23 shows that Bax increased inmitochondria and decreased in cytosol on PAE/flk-1 cells afterGrB/VEGF₁₂₁ treatment for 24 h at 20 nM concentration, indicating thatBax translocated from the cytosol to the mitochondria during apoptosis.

Example 37 Internalization of GrB/scFvMEL into A375-M Cells byImmunofluorescence Microscopy

[0431] Internalization analysis of GrB/scFvMEL by immunofluorescencemicroscopy utilized the following methods. Cells were plated in 16-wellchamber slides (Nunc), 1×10⁴ cells per well, and incubated for overnightat 37° C./5% CO₂ conditions. Cells were treated with 100 nM ofGrB/scFvMEL for 1 h and 6 h, then washed briefly with PBS. Cell surfacewas stripped by incubations of 10 min with glycine buffer (500 mM NaCl,0.1 M glycine, pH 2.5), neutralized for 2 min with 0.5 M Tris, pH 7.4,washed briefly with PBS.

[0432] For immunofluorescent staining, cells were fixed in 3.7%formaldehyde for 15 min at RT, followed by a brief rinse with PBS andthen permeabilization for 10 min in PBS containing 0.2% Triton X-100;the cells were then washed three times with PBS. Samples were incubatedwith 3% BSA for 1 h at RT to block nonspecific binding sites, thenincubated with mouse anti-granzyme B antibody (1: 100 dilution) at RTfor 1 h followed by washing three times with PBS. The samples wereincubated with fluorescein isothiocyanate (FITC)-coupled anti-mouse IgG(Sigmna) (1: 100 dilution) at RT for 1 h, washed three times with PBS.The walls and gaskets were removed carefully. After air drying, theslide was mounted and analyzed under light and fluorescence microscope.

[0433] The results showed that GrB moiety of GrB/scFvMEL was deliveredinto the gp240 antigen-positive A375-M cells by scFvMEL binding to gp240antigen.

Example 38 GrB/scFyMEL Induces Apoptosis on A375-M Cells Detected byTunel Assay

[0434] Cells (3000 cells per well) were treated with GrB/scFvMEL atI.C.₅₀ concentration for different times (16 h, 24 h) and washed brieflywith PBS. Cells were fixed with 3.7% formaldehyde at room temperaturefor 20 min, followed by rinsing with PBS and permeabilization with 0.1%Triton X-100, 0.1% sodium citrate on ice for 2 min. They were thenwashed with PBS twice. Cells were incubated with TUNEL reaction mixtureat 37° C. for 60 min. After a final wash step, the cells were analyzedunder fluorescence microscopy.

[0435] The results demonstrated that GrB/scFvMEL induced apoptosis onantigen-positive A375-M cells but not on antigen-negative SKBR3-HP cellsafter treatment for 16 h and24 h.

Example 39 Cytochrome C Release in A375-M VS. SKBr3-HP Cells Treatedwith GrB/scFvMEL

[0436] Cytochrome c release apoptosis assay was performed as described(Oncogene Research Products; San Diego, Calif.; Cat# QIA87). Thematerials and methods were as follows. Human melanoma A375-M cells andhuman breast cancer SKBR3-HP cells (5×10⁷) were treated with GrB/scFvMELat concentrations of 5 nM and 50 nM for 24 h. Cells were collected.After washing cells with 10 ml of ice-cold PBS, cells were resuspendedwith 0.5 ml of 1×cytosol extraction buffer mix containing DTT andprotease inhibitors, incubate on ice for 10 min. Cells were homogenizedin an ice-cold glass homogenizer. Homogenate was transferred to a 1.5 mlmicrocentrifuge tube, and centrifuge at 700×g for 10 min at 4° C. Thesupernatant was transferred to a fresh 1.5 ml tube, and centrifuged at10,000×g for 30 min at 4 ° C. Supernatant was collected as a cytosolicfraction. The pellet was resuspended in 0.1 ml Mitochondrial ExtractionBuffer Mix containing DTT and protease inhibitors, vortexed for 10seconds, and saved as a mitochondrial fraction. Protein concentrationswere determined by using Bio-Rad Laboratories, Inc. (Hercules, Calif.)Bradford Protein Assay. 10 μg of each cytosolic and mitochondrialfraction isolated from non-treated and treated cells were loaded on a15% SDS-PAGE, and then a standard Western blot procedure was performedand probed with cytochrome c antibody (1 μg/ml).

[0437] Cytochrome c release apoptosis assay: A375-M and SKBR3-HP cellswere treated with GrB/scFvMEL at different concentrations for 24 h. Themitochondrial fraction was isolated from the cytosol, and thencytochrome c release from mitochondria into cytosol during apoptosis wasdetermined by western blotting using cytochrome c antibody. FIG. 24indicates that cytochrome c release on A375-M but not on SKBR3-HP cellsafter GrB/scFvMEL treatment.

Example 40 GrB/VEGF₁₂₁ Induces Dna Laddering on Pae/Flk-1 CELLS

[0438] A DNA laddering assay procedure was followed to analyze influenceof GrB/VEGF121 on PAE/FLK-1 cells.

[0439] PAE cells (2×10⁶) were treated with GrB/VEGF121 at the I.C.₅₀concentration for 24 h. Cells were briefly washed with PBS and thenharvested by trypsinization, followed by centrifugation at 200×g for 5min. Cells were resuspended in 1 ml of PBS, transferred into 10 ml ofice-cold 70% ethanol and stored at −20° C. for 24 h or longer. Fixedcells were centrifuged at 800×g for 5 min, and the ethanol was removedthoroughly. The cell pellets were resuspended in 40 μl phosphate-citratebuffer (PCB), consisting of 192 parts of 0.2 M Na₂HPO₄ and 8 parts of0.1 M citric acid (pH 7.8) and incubated at RT for at least 30 min.After spinning cells down at 1000×g for 5 min, the supernatant wastransferred to a new tube. To samples were added 3 μl of 0.25% NonideNP-40 and 3 μl of RNase (1 mg/ml) and incubated for 30 min at 37° C.,followed by addition of 3 μl of proteinase K (1 mg/ml) and incubationfor another 30 min at 37° C. An 1.5% agarose gel was run to detect DNAby ethidium bromide under UV light.

[0440]FIG. 25 shows that GrB/VEGF121 induces DNA laddering on PAE/flk-1but not on PAE/flt-1 cells.

Example 41 Elisa of GrB/scFvMEL on gp240 AG-Positive A375-M VS gp240AG-Negative T-24 Cells Detected by GrB Mouse Mab

[0441] Binding activity of GrB/scFvMEL to gp240 antigen-positive humanmelanoma A375-M vs gp240 antigen-negative human bladder cancer T-24cells was analyzed by ELISA

[0442] Ninety six-well plates coated with 50,000 cells per well ofA375-M or T-24 cells were blocked by 5% BSA, and the cells were thenreacted with GrB/scFvMEL at various concentration for 1 h at RT. Afterwashing, the samples were incubated with GrB mouse monoclonal antibody(1 μg/ml) at RT for 1 h, followed by HRP-goat anti-mouse IgG. Thensubstrate 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)solution with 1 μl/ml of 30% H₂O₂ added. Absorbance at 405 nm wasmeasured after 30 min.

[0443]FIG. 26 shows that GrB/scFvMEL could specifically bind toAg-positive A375-M but not bind to Ag-negative T-24 cells, indicatingthere was binding activity of scFvMEL moiety of the fusion GrB/scFvMEL.

References

[0444] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

Patents

[0445] EPO 0273085

[0446] EPO 03089

[0447] GB 2193095A

[0448] PCT/US85/01161

[0449] PCT/US89/5040

[0450] U.S. Pat. No. 4,162,282

[0451] U.S. Pat. No. 4,215,051

[0452] U.S. Pat. No. 4,310,505

[0453] U.S. Pat. No. 4,376,110

[0454] U.S. Pat. No. 4,533,254

[0455] U.S. Pat. No. 4,554,101

[0456] U.S. Pat. No. 4,676,980

[0457] U.S. Pat. No. 4,728,575

[0458] U.S. Pat. No. 4,728,578

[0459] U.S. Pat. No. 4,737,323

[0460] U.S. Pat. No. 4,816,567

[0461] U.S. Pat. No. 4,921,706

[0462] U.S. Pat. No. 4,946,778

[0463] U.S. Pat. No. 5,401,511

[0464] U.S. Pat. No. 5,401,511

[0465] U.S. Pat. No. 5,432,260

[0466] U.S. Pat. No. 5,440,013

[0467] U.S. Pat. No. 5,446,128

[0468] U.S. Pat. No. 5,475,085

[0469] U.S. Pat. No. 5,603,872

[0470] U.S. Pat. No. 5,603,872

[0471] U.S. Pat. No. 5,618,914

[0472] U.S. Pat. No. 5,656,725

[0473] U.S. Pat. No. 5,670,155

[0474] U.S. Pat. No. 5,672,681

[0475] U.S. Pat. No. 5,674,976

[0476] U.S. Pat. No. 5,710,245

[0477] U.S. Pat. No. 5,786,214

[0478] U.S. Pat. No. 5,840,833

[0479] U.S. Pat. No. 5,849,718

[0480] U.S. Pat. No. 5,859,184

[0481] U.S. Pat. No. 5,871,727

[0482] U.S. Pat. No. 5,879,703

[0483] U.S. Pat. No. 5,889,155

[0484] U.S. Pat. No. 5,889,155

[0485] U.S. Pat. No. 5,929,237

[0486] U.S. Pat. No. 5,939,277

[0487] U.S. Pat. No. 6,107,090

[0488] U.S. patent Ser. No. 07/715,272

[0489] U.S. patent Ser. No. 07/931,811

[0490] U.S. patent Ser. No. 07/934,373

[0491] WO 91/00360

[0492] WO 92/200373

[0493] WO 93/06213

[0494] WO 93/08829

[0495] WO 97/19179

[0496] WO 97/22364

[0497] WO 97/46259

[0498] WO 98/0748

[0499] WO 99/18933

[0500] WO 99/45128

[0501] WO 99/49059

Publications

[0502] Adams G P, Schier R. Generating improved single-chain Fvmolecules for tumor targeting. J Immunol Methods. 1999 December10;231(1-2):249-60.

[0503] Adams J. M. and Cory S. The Bcl-2 protein family: arbiters ofcell survival. Science, 281:1322-1326, 1998.

[0504] Aksentijevich I, Pastan I, Lunardi-Iskandar Y, Gallo R C,Gottesman M M, Thierry A R. In vitro and in vivo liposome-mediated genetransfer leads to human MDR1 expression in mouse bone marrow progenitorcells. Hum Gene Ther. 1996 June 10;7(9):1111-22.

[0505] Andrade F, Roy S, Nicholson D, Thomberry N, Rosen A,Casciola-Rosen L. Granzyme B directly and efficiently cleaves severaldownstream caspase substrates: implications for CTL-induced apoptosis.Immunity 1998: 8: 451-460.

[0506] Antonsson B. et al. Inhibition of Bax channel-forming activity byBcl-2. Science. 277: 370-372, 1997.

[0507] Aqeilan R, Yarkoni S, Lorberboum-Galski H. Interleukin 2-Bax: anovel prototype of human chimeric proteins for targeted therapy. FEBSLett. 1999 August 27;457(2):271-6.

[0508] Arap et al., 1995.

[0509] Ashkenazi A, Dixit V M. Death receptors: signaling andmodulation. Science 1998; 281: 1305-1308.

[0510] Ausubel et al., In: Current Protocols in Molecular Biology, John,Wiley & Sons, Inc., 1994.

[0511] Bangham, et al., “Diffusion of univalent Ions across the Lamellaeof Swollen Phospholipids” J. Mol. Biol., 13:238-252, 1965.

[0512] Barclay et al. (eds.), The Leucocyl=te Antigen Facts Book, 1993,Academic Press.

[0513] Becker K G, Mattson D H, Powers J M, Gado A M, Biddison W E.Analysis of a sequenced cDNA library from multiple sclerosis lesions. JNeuroimmunol. 1997 July;77(1):27-38.

[0514] Beresford P J. Xia Z, Greenberg A H, Lieberman J. Granzyme Aloading induces rapid cytolysis and a novel from of DNA damageindependently of caspase activation. Immunity 1999; 10: 585-594.

[0515] Berke G. The CTL's kiss of death, Cell 1995; 81: 9-12.

[0516] Bird, R. E. et al., Science 242, 423-426, 1988.

[0517] Boise L H, Gonzalez-Garcia M, Postema C E, Ding L, Lindsten T,Turka L A, Mao X, Nunez G, Thompson C B. bcl-x, a bcl-2-related genethat functions as a dominant regulator of apoptotic cell death. Cell.1993 August 27;74(4):597-608.

[0518] Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. Involvement ofMACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1-and TNFreceptor-induced cell death. Cell 1996; 85: 803-815.

[0519] Boyd J M, Gallo G J, Elangovan B, Houghton A B, Malstrom S, AveryB J, Ebb R G, Subramanian T, Chittenden T, Lutz R J, et al. Bik, a noveldeath-inducing protein shares a distinct sequence motif with Bcl-2family proteins and interacts with viral and cellular survival-promotingproteins. Oncogene. 1995 November 2;1 1(9):1921-8.

[0520] Brinkmann U, Di Carlo A, Vasmatzis G, Kurochkina N, Beers R, LeeB, Pastan I. Stabilization of a recombinant Fv fragment by base-loopinterconnection and V(H)-V(L) permutation. J Mol Biol. 1997 April25;268(1):107-17.

[0521] Brodeur et al., Monoclonal Antibody Production Techniques &Applications, pp. 51-63 (Marcel Dekker Inc., New York, 1987).

[0522] Browne, K. A., Blink, E., Sutton, V. R., Groelich, C. J., Jans,D. A., and Trapani, J. A. Mol. Cell. Biol. 1999; 19: 8604-8615.

[0523] Canfield L M, Fritz T A, Tarara T E. Incorporation ofbeta-carotene into mixed micelles. Methods Enzymol. 1990;189:418-22.

[0524] Caruthers M H, Beaucage S L, Efcavitch J W, Fisher E F, MatteucciM D, Stabinsky Y. New chemical methods for synthesizing polynucleotides.Nucleic Acids Symp Ser. 1980;(7):215-23.

[0525] Catalysis in Micellar & Macromolecular Systems, 1975.

[0526] Cheng J Q, Jhanwar S C, Klein W M, Bell D W, Lee W C, Altomare DA, Nobori T, Olopade O I, Buckler A J, Testa J R. p 16 alterations anddeletion mapping of 9p21-p22 in malignant mesothelioma. Cancer Res. 1994Nov 1;54(21):5547-51.

[0527] Chinnaiyan A. M., Orth K., O'Rourke K., Duan, H., Poirier G. G.and Dixit, V. M. Molecular ordering of the cell death pathway. J. Biol.Chem., 271: 4573-4576, 1996.

[0528] Chinnaiyan A M, Orth K, Hanna W L, Duan H J, Poirier G G,Froelich C J, Dixit V M, Cytotoxic T cell-derived granzyme B activatesthe apoptotic protease ICE-LAP3. Curr Biol 1996; 6: 897-899.

[0529] Clackson T, Hoogenboom H R, Griffiths A D, Winter G. Makingantibody fragments using phage display libraries. Nature. 1991 August15;352(6336):624-8.

[0530] Cleary M L, Sklar J. Nucleotide sequence of a t(14; 18)chromosomal breakpoint in follicular lymphoma and demonstration of abreakpoint-cluster region near a transcriptionally active locus onchromosome 18. Proc Natl Acad Sci U S A. 1985 November;82(21):7439-43.

[0531] Cohen, 1997, Biochem. J. 326:1-16.

[0532] Colbere-Garapin F, Horodniceanu F, Kourilsky P, Garapin A C. Anew dominant hybrid selective marker for higher eukaryotic cells. J MolBiol. 1981 July 25; 150(1):1-14.

[0533] Cole S P, Vreeken E H, Roder J C. Antibody production by human Xhuman hybridomas in serum-free medium. J Immunol Methods. 1985 April22;78(2):271-8.

[0534] Colloidal Surfactant, 1963.

[0535] Creighton, 1983, Proteins Structures & Molecular Principles, W.H. Freeman & Co., N. Y., pp. 34-60.

[0536] Darmon A J, Ley T J, Nicholson D W, Bleackley R C, Cleavage ofCPP32 by granzyme B represents a critical role for granzyme B in theinduction of target cell DNA fragmentation. J Biol Chem 1996; 271:21709-21712.

[0537] Darmon A J, Nicholson D W, Bleackley R C. Activation of theapoptotic protease CPP32 by cytotoxic T-cell derived granzyme B. Nature1995; 377: 446-448.

[0538] Deamer and P. Uster, “Liposome Preparation: Methods andMechanisms,” in Liposomes (M. Ostro, ed.), Marcel Dekker, Inc., New York(1983), pp. 27-52.

[0539] Doherty P C. Cell-mediated cytotoxicity. Cell 1993; 75: 607-612.

[0540] Duan J, Orth K, Chinnaiyan A M, Poirier G G, Froelich C J, HeW-W, Dixit V M. ICE-LAP6, a novel member of the ICE/Ced-3 gene family,is activated by the cytotoxic T cell protease granzyme B. J Biol Chem1996; 271: 16720-16724.

[0541] Ebnet K, Hausmann M, Lehmann-Grube F, Mullbacher A, Kopf M,Lamers M, Simon M M, Granzyme A-deficient mice retain potentcell-mediated cytotoxicity. EMBO J 1995; 14: 4230-4239.

[0542] El-Gorab M. Solubilization of -carotene and retinol into aqueoussolutions of mixed micelles.Biochim Biophys Acta. 1973April13;306(1):58-66.

[0543] Felgner P L, Gadek T R, Holm M, Roman R, Chan H W, Wenz M,Northrop J P, Ringold G M, Danielsen M. Lipofection: a highly efficient,lipid-mediated DNA-transfection procedure.Proc Natl Acad Sci U S A. 1987November;84(21):7413-7.

[0544] Fernandes-Alnemri T, Armstrong R C, Krebs J, Srinivasula S M,Wang L, Bullrich F, Fritz L C, Trapani J A, Tomaselli K J, Litwack G,Alnemri E S. In vitro activation of CPP32 and Mch3 by Mch4, a novelhuman apoptotic cysteine protease containing two FADD-like domains.Proc. Natl Acad Sci USA 1996; 93: 7464-7469.

[0545] Fernandes-Alnemri T, Litwack G, Alnemri S. Mch2, a new member ofthe apoptotic Ced-3/Ice cysteine protease gene family. Cancer Res 1995;55: 2737-2742.

[0546] FernandeS-Alnemri T, Takahashi A, Armstrong R, Krebs J, Fritz L,Tomaselli K J, Wang L, Yu Z, Croce C M, Salvesen G, Earnshaw W C,Litwack G, Alnemri E S. Mch3, a novel human apoptotic cysteine proteasehighly related to CPP32. Cancer Res 1995; 55: 6045-6052.

[0547] Fingl et al., 1975, In: The Pharmacological Basis ofTherapeutics, Ch. 1, p. 1.

[0548] Fraley and Fornari Kaplan, “Entrapment of a bacterial plasmid inphospholipid vesicles:potential for gene transfer,” Proc. Nat'l. Acad.Sci. USA 76:3348-3352, 1979.

[0549] Froelich C J, Orth K, Turbov J, Seth P, Gottlieb R, Babior B,Shah G M, Bleackley R C, Dixit V M, Hanna W. New paradigm for lymphocytegranule-mediated cytotoxicity. Target cells bind and internalizegranzyme B, but an endosomolytic agent is necessary for cytosolicdelivery and subsequent apoptosis. J Biol Chem 1996; 271: 29073-29079.

[0550] Gazzaniga P, Gradilone A, Vercillo R, Gandini O, Silvestri I,Napolitano M, Albonici L, Vincenzoni A, Gallucci M, Frati L, Agliano AM. Bcl-2/bax mRNA expression ratio as prognostic factor in low-gradeurinary bladder cancer. Int J Cancer. 1996 Apr 22;69(2): 100-4.

[0551] Gershenfeld H K, Weissman I L. Cloning of cDNA for a Tcell-specific serine protease from a cytotoxic T lymphocyte. Science1986; 232: 854-858.

[0552] Ghosh and Bachhawat, “Targeting of liposomes to hepatocytes,” In:Wu G. Wu C ed., Liver diseases, targeted diagnosis and therapy usingspecific receptors and ligands, New York: Marel Dekker, pp. 87-104,1991.

[0553] Goding, Monoclonal Antibodies: Principles & Practice, pp. 59-104(Academic Press, 1986).

[0554] Gregoriadis G. and Davis C. “Stability of liposomes in vivo andin vitro is promoted by their cholesterol content and the presence ofblood cells,” Biochem Biophys Res Commun., 89(4):1287-1293, 1979.

[0555] Gregoriadis, DRUG CARRIERS IN BIOLOGY AND MEDICINE, G.Gregoriadis (ed.), 1979, pp. 287-341.

[0556] Griffiths, G. M., and 1saaz, S. J. Cell Biol. 1993; 120: 885-896.

[0557] Gross A., Jockel J., Wie M. C. and Korsmeyer S. J. Enforceddimerization of Bax results in its translocation, mitochondrialdysfuinction and apoptosis. EMBO J. 17: 3878-3885, 1998.

[0558] Gu Y, Samecki C, Fleming M A, Lippke J A, Bleackley R C, Su MS-S.Processing and activation of CMH-1 by granzyme B. J Biol Chem 1996; 271:10816-10820.

[0559] Haddad, P., Jenne, D., Tschopp, J., Clement, M. V.,Mathieu-Mahul, D., and Sasportes, M. Int. Immunol. 1991; 3: 57-66.

[0560] Hayes M P, Berrebi G A, Henkart P A. Induction of target cell DNArelease by the cytotoxic T lymphocyte granule protease granzyme.A. J ExpMed 1980; 170: 933-946.

[0561] Heath et al., Chem. Phys. Lipids 40:347 (1986).

[0562] Heibein, J. A., Barry, M., Motyka, B., and Bleakley, R. C. J.Immunol. 1999; 163: 4683-4693.

[0563] Henkart, P. A. Annu. Rev. Immunol. 1985; 3: 31-58.

[0564] Heusel J W, Wesselschmidt R L, Shresta S, Russell J H, Ley T J.Cytotoxic lymphocytes require granzyme B for the rapid induction of DNAfragmentation and apoptosis in allogeneic target cells. Cell 1994; 76:977-987.

[0565] Hockenbery D. M., Oltvai Z. N., Yin X.-M., Milliman C. L. andKorsmeyer S. J. Bcl-2 functions in an antioxidant pathway to preventapoptosis. Cell, 75: 241-251, 1993.

[0566] Hollstein M, Sidransky D, Vogelstein B, Harris C C. p53 mutationsin human cancers. Science. 1991 July 5;253(5015):49-53.

[0567] Huston, J. S. et al., Proc. Natl. Acad. Sci., USA 85, 5879-5883(1988).

[0568] Hsu Y. T. and Youle R. J. Nonionic detergents induce dimerizationamong members of the Bcl-2 family. J. Biol. Chem., 272: 13829-13834,1997.

[0569] Huiling H., Lam M., McCormic T. S. and Distelhorst C. W.Maintenance of calcium homeostasis in the endoplasmic reticulum byBcl-2. J. Cell Biol. 138: 1219-1228, 1997.

[0570] Hussussian C J, Struewing J P, Goldstein A M, Higgins P A, Ally DS, Sheahan M D, Clark W H Jr, Tucker M A, Dracopoli N C. Germline p16mutations in familial melanoma. Nat Genet. 1994 September;8(1):15-21.

[0571] Huston J S, Levinson D, Mudgett-Hunter M, Tai M S, Novotny J,Margolies M N, Ridge R J, Bruccoleri R E, Haber E, Crea R, et al.Protein engineering of antibody binding sites: recovery of specificactivity in an anti-digoxin single-chain Fv analogue produced inEscherichia coli. Proc Natl Acad Sci U S A. 1988 August;85(16):5879-83.

[0572] Inohara N, Ding L, Chen S, Nunez G. harakiri, a novel regulatorof cell death, encodes a protein that activates apoptosis and interactsselectively with survival-promoting proteins Bcl-2 and Bcl-X(L). EMBO J.1997 April 1;16(7):1686-94.

[0573] Jacobson M. D., Weil M, and Raff M. C. Programmed cell death inanimal development. Cell, 88: 347-354, 1997.

[0574] Jakobovits A, Moore A L, Green L L, Vergara G J, Maynard-Currie CE, Austin H A, Klapholz S. Germ-line transmission and expression of ahuman-derived yeast artificial chromosome. Nature. 1993 March18;362(6417):255-8.

[0575] Jans D A, Jans P, Briggs L J, Sutton V, Trapani J A. Nucleartransport of granzyme B (fragmentin-2). J Biol Chem 1996; 271:30781-30789.

[0576] Johannesson et al., 1999, “Bicyclic tripeptide mimetics withreverse turn inducing properties.” J Med. Chem. 42:601-608.

[0577] Jones P T, Dear P H, Foote J, Neuberger M S, Winter G. Replacingthe complementarity-determining regions in a human antibody with thosefrom a mouse. Nature. 1986 May 29-June 4;321(6069):522-5.

[0578] Kagawa S., Gu J., Swisher S. G., Ji L., RothJ. A. Lai D.,Stephens L. C. and Fang B. Antitumor effect of adenovirus-mediated Baxgene transfer on p53-sensitive and p53-resistant cancer lines. CancerResearch. 60: 1157-1161, 2000.

[0579] Kagawa S., Pearson S. A., Ji L., Xu K., McDonnell T. J., SwisherS. G., Roth J. A. and Fang B. A binary adenoviral vector system forexpressing high levels of the proapoptotic gene bax. Gene Therapy., 7:75-79, 2000.

[0580] Kagi D, Lederman B. Burki K, Zinkernagel R M, Hengartner H.Molecular mechanisms of lymphocyte-mediated cytoxicity and their role inimmunological protection and pathogenesis in vivo. Ann Rev Immunol 1996;14:207-232.

[0581] Kam, C-M., Hudig D., and Powers, J. C. Biochimica et BiophysicaActa. 2000; 1477: 307-323.

[0582] Kamb A, Shattuck-Eidens D, Eeles R, Liu Q, Gruis N A, Ding W,Hussey C, Tran T, Miki Y, Weaver-Feldhaus J, et al. Analysis of the p16gene (CDKN2) as a candidate for the chromosome 9p melanomasusceptibility locus. Nat Genet. 1994 September; 8(1):23-6.

[0583] Kaneda et al., “Increased expression of DNA cointroduced withnuclear protein in adult rat liver,” Science, 243:375-378, 1989.

[0584] Kaneda et al., “Introduction and expression of the human insulingene in adult rat liver,” J Biol Chem., 264(21):12126-12129, 1989.

[0585] Kato et al., “Expression of hepatitis B virus surface antigen inadult rat liver. Co-introduction of DNA and nuclear protein by asimplified liposome method,” J Biol Chem., 266(6):3361-3364, 1991.

[0586] Kerr J F, Wyllie A H, Currie A R. Apoptosis: a basic biologicalphenomenon with wide-ranging implications in tissue kinetics. Br JCancer. 1972 August;26(4):239-57.

[0587] Kiefer M C, Brauer M J, Powers V C, Wu J J, Umansky S R, Tomei LD, Barr P J. Modulation of apoptosis by the widely distributed Bcl-2homologue Bak. Nature. 1995 April 20;374(6524):736-9.

[0588] Knudson C M, Korsmeyer S J. Bcl-2 and Bax function independentlyto regulate cell death. Nat Genet. 1997 August; 16(4):358-63.

[0589] Kozbor D, Tripputi P, Roder J C, Croce C M. A human hybridmyeloma for production of human monoclonal antibodies. J Immunol. 1984December;133(6):3001-5.

[0590] Kozopas K M, Yang T, Buchan H L, Zhou P, Craig R W. MCL1, a geneexpressed in programmed myeloid cell differentiation, has sequencesimilarity to BCL2. Proc Natl Acad Sci U S A. 1993 April15;90(8):3516-20.

[0591] Kuroki M, Arakawa F, Khare P D, Kuroki M, Liao S, Matsumoto H,Abe H, Imakiire T. Specific targeting strategies of cancer gene therapyusing a single-chain variable fragment (scFv) with a high affinity forCEA. Anticancer Res. 2000 November-December;20(6A):4067-71.

[0592] Kyte & Doolittle, 1982.

[0593] Lam M., Dubyak G., Chen L., Nunez G., Miesfeld R. L. andDistelhorst C. W. Evidence that Bcl-2 represses apoptosis by regulatingendoplasmic reticulum-associated Ca2+ fluxes. Proc. Natl. Acad. Sci.USA, 91: 6569-6573, 1994.

[0594] Lang J, Vigo-Pelfrey C, Martin F. Liposomes composed of partiallyhydrogenated egg phosphatidylcholines: fatty acid composition, thermalphase behavior and oxidative stability. Chem Phys Lipids. 1990March;53(1):91-101.

[0595] Lin E Y, Orlofsky A, Berger M S, Prystowsky M B. Characterizationof A1, a novel hemopoictic-specific early-response gene with sequencesimilarity to bcl-2. J Immunol. 1993 August 15;151(4):1979-88.

[0596] Liposome Technology, 1984.

[0597] Liu Y, Liggitt D, Zhong W, Tu G, Gaensler K, Debs R. Cationicliposome-mediated intravenous gene delivery. J Biol Chem. 1995 October20;270(42):24864-70.

[0598] Liu X, Kim C N, Pohl J, Wang X. Purification and characterizationof an interleukin-1beta-converting enzyme family protease that activatescysteine protease P32 (CPP32). J Biol Chem. 1996 June 7;271(23):13371-6.

[0599] Liu X, Zou H, Slaughter C, Wang X. D F F, a heterodimeric proteinthat functions downstream of caspase-3 to trigger DNA fragmentationduring apoptosis. Cell. 1997 April 18;89(2): 175-84.

[0600] Lobe C G, Havele C, Bleackley R C. Cloning of two genes that arespecifically expressed in activated cytotoxic T lymphocytes. Proc NatlAcad Sci USA 1986; 83: 1448-1452.

[0601] Lowy I, Pellicer A, Jackson J F, Sim G K, Silverstein S, Axel R.Isolation of transforming DNA: cloning the hamster aprt gene. Cell. 1980December; 22(3):817-23.

[0602] Marks J D, Hoogenboom H R, Bonnert T P, McCafferty J, Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-genelibraries displayed on phage. J Mol Biol. 1991 December 5;222(3):581-97.

[0603] Marks J D, Griffiths A D, Malmqvist M, Clackson T P, Bye J M,Winter G. By-passing immunization: building high affinity humanantibodies by chain shuffling. Biotechnology (N Y). 1992Jul;10(7):779-83.

[0604] Martin S J, Amarante-Mendes G P, Shi L F, Chuang T H, Casiano CA, O'Brien G A, Fitzgerald P, Tan E M, Bokoch G M, Greenberg A H, GreenD R. The cytotoxic cell protease granzyme B initiates apoptosis in acell-free system by proteolytic processing and activation of theICE/CED-3 family protease, CPP32, via a novel two-step mechanism. EMBO J1996: 15: 2407-2416.

[0605] Marzo I, et al. The permeability transition pore complex: atarget for apoptosis regulation by caspases and bcl-2-related proteins.J. Exp. Med. 187: 1261-1271, 1998.

[0606] Masson D, Tschopp J. Isolation of a lytic pore-forming protein(perforin) from cytolytic T lymphocytes. J Biol Chem 1985;260:9069-9072.

[0607] Masson D, Zamai M, Tschopp J. Identification of granzyme Aisolated from cytotoxic T-lymphocyte-granules as one of the proteasesencoded by CTL-specific genes. FEBS Lett 1986; 208: 84-88.

[0608] Mayer L D, Hope M J, Cullis P R. Vesicles of variable sizesproduced by a rapid extrusion procedure. Biochim Biophys Acta. 1986 June13;858(1):161-8.

[0609] Mayhew E, Conroy S, King J, Lazo R, Nikolopoulus G, Siciliano A,Vail W J. High-pressure continuous-flow system for drug entrapment inliposomes. Methods Enzymol. 1987;149:64-77.

[0610] McConlogue, L., 1987, In: Current communications in molecularbiology, Cold Spring Harbor Lab ed.)

[0611] Medema J P, Toes R E M, Scaffidi C, Zheng T S, Flavell R A,Melief C J M, Peter M E, Offringa R, Krammer P H. Cleavage of FLICE(caspase-8) by granzyme B during cytotoxic T lymphocyte-inducedapoptosis. Eur J Immunol 1997; 27: 3492-3498.

[0612] Milstein C, Cuello AC. Hybrid hybridomas and their use inimmunohistochemistry. Nature. 1993 October 6-12;305(5934):537-40.

[0613] Modem Pharmaceutics, 1990.

[0614] Montaldo P G, Pagnan G, Pastorino F, Chiesa V, Raffaghello L,Kirchmeier M, Allen T M, Ponzoni M. N-(4-hydroxyphenyl) retinamide iscytotoxic to melanoma cells in vitro through induction of programmedcell death. Int J Cancer. 1999 April 12;81(2):262-7.

[0615] Morrison S L, Johnson M J, Herzenberg L A, Oi V T. Chimeric humanantibody molecules: mouse antigen-binding domains with human constantregion domains. Proc Natl Acad Sci U S A. 1984November; 81(21):6851-5.

[0616] Mujoo K, Spiro R C, Reisfeld R A. Characterization of a uniqueglycoprotein antigen expressed on the surface of human neuroblastomacells. J Biol Chem. 1986 August 5:261(22): 10299-305.

[0617] Mullbacher A, Ebnet K, Blanden R V, Hla R T, Stehle T, MuseteanuC, Simon M M. Granzyme A is critical for recovery of mice from infectionwith the natural cytopathic viral pathogen, ectromelia. Proc Natl AcadSci USA 1996; 93: 5783-5787.

[0618] Mulligan R C, Berg P. Factors governing the expression of abacterial gene in mammalian cells. Mol Cell Biol. 1981 May; 1(5):449-59.

[0619] Muzio M, Chinnaiyan A M, Kischkel F C, O'Rourke K, Shevchenko A,Ni J, Scaffidi C, Bretz J D, Zhang M, Gentz R, Mann M, Krammer P H,Peter M E, Dixit V M. FLICE, a novel FADD-homologous ICE/CED-3-likeprotease, is recruited to the CD95 (Fas/APO-1) death-inducing signalingcomplex. Cell 1996; 85: 817-827.

[0620] Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K.,Shevchenko, A., Scaffidi, C., Bretz, J. D., Zhang, M., Ni, J., Gentz,R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. Cell. 1996;86: 817-821.

[0621] Nagata S, Golstein P. The Fas death factor. Science 1995; 267:1449-1456.

[0622] Nechushtan A, Yarkoni S, Marianovsky I, Lorberboum-Galski H.Adenocarcinoma cells are targeted by the new GnRH-PE66 chimeric toxinthrough specific gonadotropin-releasing hormone binding sites. J BiolChem. 1997 April 25;272(17):11597-603.

[0623] Neuberger M S, Williams G T, Fox R O. Recombinant antibodiespossessing novel effector functions. Nature. 1

[0624]4-8.

[0625] Nicolau and Sene, “Liposome-mediated DNA transfer in eukaryoticcells: dependence of the transfer efficiency upon the type of liposomesused and the host cell cycle stage,” Biochem. Biophys. Acta, 721:185-1901982.

[0626] Nicolau et al., “Liposomes as carriers for in vivo gene transferand expression,” Methods Enzymol., 149:157-176, 1987.

[0627] Nygren H. Conjugation of horseradish peroxidase to Fab fragmentswith different homobifunctional and heterobifunctional cross-linkingreagents. A comparative study. J Histochem Cytochem. 1982 May;30(5):407-12.

[0628] O'Hare K, Benoist C, Breathnach R. Transformation of mousefibroblasts to methotrexate resistance by a recombinant plasmidexpressing a prokaryotic dihydrofolate reductase. Proc Natl Acad Sci U SA. 1981 March; 78(3):1527-31.

[0629] Odake S, Kam C M, Narasimhan L, Poe M, Blake J T, Krahenbuhl O,Tschopp J, Powers J C. Human and murine cytotoxic T lymphocyte serineproteases: subsite mapping with peptide thioester substrates andinhibition of enzyme activity and cytolysis by isocoumarins. Biochem1991; 30: 2217-2227.

[0630] Oltvai Z. N., Milliman C. L. and Korsmeyer S. J. Bcl-2heterodimerizes in vivo with a conserved homolog, Bax, that acceleratesprogrammed cell death. Cell, 74: 609-619, 1993.

[0631] Orth K, Chinnaiyan A M, Garg M, Froelich C J, Dixit V M. TheCED-3/ICE-like protease Mch2 is activated during apoptosis and cleavesthe death substrate lamin A. J. Biol Chem 1996; 271: 16443-16446.

[0632] Ottilie S, Diaz J L, Home W, Chang J, Wang Y, Wilson G, Chang S,Weeks S, Fritz L C, Oltersdorf T. Dimerization properties of human BAD.1dentification of a BH-3 domain and analysis of its binding to mutantBCL-2 and BCL-XL proteins. J Biol Chem. 1997 December5;272(49):30866-72.

[0633] Pagnan G, Montaldo P G, Pastorino F, Raffaghello L, Kirchmeier M,Allen T M, Ponzoni M. GD2-mediated melanoma cell targeting andcytotoxicity of liposome-entrapped fenretinide. Int J Cancer. 1999 April12;81(2):268-74.

[0634] Perales et al., “Gene transfer in vivo: sustained expression andregulation of genes introduced into the liver by receptor-targeteduptake,” Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994.

[0635] Pinkoski M J, Heibein J A, Barry M, Bleackley R C. Nucleartranslocation of granzyme B in target cell apoptosis. Cell Death Differ2000; 7: 17-24.

[0636] Pinkoski M J, Hobman M, Helbein J A, Tomaselli K, Li F, Seth P,Froelich C J, Bleackley R C. Entry and trafficking of granzyme B intarget cells during granzyme B-perforin-mediated apoptosis. Blood1998;92:1044-1054.

[0637] Pinkoski M J, Winkler U, Hudig D, Bleackley R C. Biding ofgranzyme B in the nucleus of target cells. Recognition of an80-kilodalton protein. J Biol Chem 1996:271:10225-10229.

[0638] Podack, E. R. Curr. Top. Microbiol. Immunol. 1992; 178: 175-184.

[0639] Poe M, Blake J T, Boulton D A, Gammon M, Sigal N H, Wu J K,Zweerink H J. Human cytotoxic lymphocyte granzyme B: its purificationfrom granules and the characterization of substrate and inhibitorspecificity. J Biol Chem 1991; 266: 98-103.

[0640] Quan L T, Tewari M, O'Rourke K, Dixit V M, Snipas S J, Poirier GG, Ray C, Pickup D J, Salvesen G S, Proteolytic activation of the celldeath protease Yama/CPP32 by granzyme B. Proc Natl Acad Sci USA 1996;93: 1972-1976.

[0641] Remington's Pharmaceutical Sciences, 18th ED. Mack PrintingCompany, 1990.

[0642] Riechmann L, Clark M, Waldmann H, Winter G. Reshaping humanantibodies for therapy. Nature. 1988 March 24;332(6162):323-7.

[0643] Ruther U, Muller-Hill B. Easy identification of cDNA clones. EMBOJ. 1983;2(10):1791-4.

[0644] Sambrook et al., 1989.

[0645] Santerre R F, Allen N E, Hobbs J N Jr, Rao R N, Schmidt R J.Expression of prokaryotic genes for hygromycin B and G418 resistance asdominant-selection markers in mouse L cells. Gene. 1984 October;30(1-3): 147-56.

[0646] Sarin, A., Williams, M. S., Alexander-Miller, M. A., Berzofsky,J. A., Zacharchuk, C. M., and Henkart, P. A. Immunity. 1997; 6: 209-215.

[0647] Schendel S L, Xie Z, Montal M O, Matsuyama S, Montal M, Reed J C.Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci U SA. 1997 May 13;94(10):5113-8.

[0648] Schmid, J., and Weissman, C. J. Immunol. 1987; 139: 250-254.

[0649] Schulz G, Cheresh D A, Varki N M, Yu A, Staffileno L K, ReisfeldR A. Detection of ganglioside GD2 in tumor tissues and sera ofneuroblastoma patients. Cancer Res. 1984 December; 44(12 Pt 1):5914-20.

[0650] Serrano M, Hannon G J, Beach D. A new regulatory motif incell-cycle control causing specific inhibition of cyclin D/CDK4. Nature.1993 December 16;366(6456):704-7.

[0651] Serrano M, Gomez-Lahoz E, DePinho R A, Beach D, Bar-Sagi D.Inhibition of ras-induced proliferation and cellular transformation byp6INK4. Science. 1995 January 13;267(5195):249-52.

[0652] Shi L, Chen G, MacDonald G, Bergeron L, Li H, Miura M, Rotello RJ, Miller D K, Li P, Seshadri T, Yuan J, Greenberg A H. Activation of aninterleukin 1 converting enzyme-dependent apoptosis pathway by granzymeB. Proc Natl Acad Sci USA 1996; 93: 11002-11007.

[0653] Shi L, Kraut R P, Aebersold R, Greenberg A H. A natural killercell granule protein that induces DNA fragmentation and apoptosis. J ExpMed 1992; 175: 553-566.

[0654] Shi L, Mai S, Israels S, Browne K, Trapani J A, Greenberg A H.Granzyme B (GraB) autonomously crosses the cell membrane and perforininitiates apoptosis and GraB nuclear localization. J Exp Med 1997; 185:853-866.

[0655] Shinkai, Y., Takio, K., and Okumura, K. Nature. 1988; 334:525-527.

[0656] Shresta S, Graubert T A, Thomas D A, Raptis S Z, Ley T J.Granzyme A initiates an alternative pathway for granule-mediatedapoptosis. Immunity 1999; 10: 595-605.

[0657] Shresta S, Heusel J W, Maclvor D M, Wesselschmidt R L, Russell JH, Ley T J. Granzyme B plays a critical role in cytotoxiclymphocyte-induced apoptosis. Immunol Rev 1995; 146: 211-221.

[0658] Shresta S, Maclvor D M, Heusel J W, Russell J H, Ley T J. Naturalkiller and lymphokine-activated killer cells require granzyme B for therapid induction of apoptosis in susceptible target cells. Proc Natl AcadSci USA 1995; 92: 5679-5683.

[0659] Smyth MJ, O'Connor M D, Trapani J A. Granzymes: A variety ofserine protease specificities encoded by genetically distinctsubfamilies. J Leuk Biol 1996; 60: 555-562.

[0660] Smyth, M. J., and Trapani, J. A. Immunol. Today. 1995; 16:202-206.

[0661] Smyth, M. J., McGuire, M. J., and Thia, K. Y. J. Immunol. 1995;154: 6299-6305.

[0662] Solodin I, Brown C S, Bruno M S, Chow C Y, Jang E H, Debs R J,Heath T D. A novel series of amphiphilic imidazolinium compounds for invitro and in vivo gene delivery. Biochemistry. 1995 October17;34(41):13537-44.

[0663] Spanjer H H, Scherphof G L. Targeting oflactosylceramide-containing liposomes to hepatocytes in vivo. BiochimBiophys Acta. 1983 September 21;734(1):40-7.

[0664] Suresh M R, Cuello A C, Milstein C. Advantages of bispecifichybridomas in one-step immunocytochemistry and immunoassays. Proc NatlAcad Sci U S A. 1986 October;83(20):7989-93.

[0665] Suzuki T, Nishiyama K, Yamamoto A, Inazawa J, Iwaki T, Yamada T,Kanazawa I, Sakaki Y. Molecular cloning of a novel apoptosis-relatedgene, human NapI (NCKAP1), and its possible relation to Alzheimerdisease. Genomics. 2000 January 15;63(2):246-54.

[0666] Szoka and Papahadjopoulos, “Procedure for Preparation ofLiposomes With Large Internal Aqueous Space. . . . ”, Proc. NatL. Acad.Sci., 75:4194-4198, 1978.

[0667] Tai Y-T, Strobel T., Kufe D., and Cannistra S. A. In vivocytotoxicity of ovarian cancer cells through tumor-selective expressionof the Bax gene. Cancer Research. 59: 2121-2126, 1999.

[0668] Takeda S, Naito T, Hama K, Noma T, Honjo T. Construction ofchimaeric processed immunoglobulin genes containing mouse variable andhuman constant region sequences. Nature. 1985 April4-10;314(6010):452-4.

[0669] Talanian R V, Yang X H, Turbov J, Seth P, Ghayur T, Casiano C A,Orth K, Froelich C J. Granule-mediated killing: pathways for granzymeB-initiated apoptosis. J Exp Med 1997; 186: 1323-1331.

[0670] Templeton N S, Lasic D D, Frederik P M, Strey H H, Roberts D D,Pavlakis G N. Improved DNA: liposome complexes for increased systemicdelivery and gene expression. Nat Biotechnol. 1997 July; 15(7):647-52.

[0671] Thierry A R, Lunardi-Iskandar Y, Bryant J L, Rabinovich P, GalloR C, Mahan L C. Systemic gene therapy: biodistribution and long-termexpression of a transgene in mice. Proc Natl Acad Sci U S A. 1995October 10;92(21):9742-6.

[0672] Thompson (ed.) 1994, The Cytokine Handbook, Academic Press, SanDiego.

[0673] Trapani J A, Browne K A, Smyth M J, Jans D A. Localization ofgranzyme B in the nucleus. A putative role in the mechanism of cytotoxiclymphocyte-mediated apoptosis. J Biol Chem 1996; 271: 4127-4133.

[0674] Trapani, J. A., Jans, D. A., Browne, K. A., Smyth, M. J., Jans,P., and Sutton, V. R. J. Biol. Chem. 1998; 273: 27934-27938.

[0675] Trapani, J. A., Klein, J., White, P. C., and Dupont, B. Proc.Natl. Acad. Sci. U.S.A. 1988; 85: 6924-6928.

[0676] Traunecker A, Lanzavecchia A, Karjalainen K. Bispecific singlechain molecules (Janusins) target cytotoxic lymphocytes on HIV infectedcells. EMBO J. 1991 December;10(12):3655-9.

[0677] Tschopp J, Schafer S, Masson D, Peitsch M C, Heusser C.Phosphorylcholine acts as a calcium dependent receptor molecule forlymphocyte perforin. Nature 1989; 337: 272-274.

[0678] Tsujimoto Y, Cossman J, Jaffe E, Croce C M. Involvement of thebcl-2 gene in human follicular lymphoma. Science. 1985 June21;228(4706):1440-3.

[0679] Tsukamoto M, Ochiya T, Yoshida S, Sugimura T, Terada M. Genetransfer and expression in progeny after intravenous DNA injection intopregnant mice. Nat Genet. 1995 March;9(3):243-8.

[0680] Van de Craen M, Van den brande I, Declercq W, Irmler M, BeyaertR, Tschopp J, Fiers W, Vandenabeele P. Cleavage of caspase familymembers by granzyme B: a comparative study in vitro. Eur J Immunol 1997;27: 1296-1299.

[0681] Van Heeke G, Schuster S M. Expression of human asparaginesynthetase in Escherichia coli. J Biol Chem. 1989 April5;264(10):5503-9.

[0682] Vaux D. L., Heacher G, and Strasser A. An evolutionaryperspective on apoptosis. Cell, 76: 777-779, 1994.

[0683] Verhoeyen M, Milstein C, Winter G. Reshaping human antibodies:grafting an antilysozyme activity. Science. 1988 March25;239(4847):1534-6

[0684] Vincenz, C., and Dixit, V. M. J. Biol. Chem. 1997; 272:6578-6583.

[0685] Vita et al., 1998, “Novel miniproteins engineered by the transferof active sites to small natural scaffolds.” Biopolymers 47:93-100.

[0686] Wagner et al., Science, 260:1510-1513, 1990.

[0687] Wang K, Yin X M, Chao D T, Milliman C L, Korsmeyer S J. BID: anovel BH3 domain-only death agonist. Genes Dev. 1996 November15;10(22):2859-69.

[0688] Waterhouse P, Griffiths A D, Johnson K S, Winter G. Combinatorialinfection and in vivo recombination: a strategy for making large phageantibody repertoires. Nucleic Acids Res. 1993 May 11;21(9):2265-6.

[0689] Weinberg R A. Tumor suppressor genes. Science. 1991 November22;254(5035):1138-46.

[0690] Weisshoff et al., 1999, “Mimicry of beta II′-turns of proteins incyclic pentapeptides with one and without D-amino acids.” Eur. J.Biochem. 259:776-788.

[0691] Wigler M, Silverstein S, Lee L S, Pellicer A, Cheng Y, Axel R.Transfer of purified herpes virus thymidine kinase gene to culturedmouse cells. Cell. 1977 May;1 1(1):223-32.

[0692] Wigler M, Perucho M, Kurtz D, Dana S, Pellicer A, Axel R,Silverstein S. Transformation of mammalian cells with an amplifiabledominant-acting gene. Proc Natl Acad Sci U S A. 1980 June;77(6):3567-70.

[0693] Wolter K. G., Hsu Y-T., Smith C. L., Nechushtan A., Xi, X.-G. andYoule R. J. Movement of Bax from the cytosol to mitochondria duringapoptosis. J. Cell Biol. 139: 1281-1292, 1997.

[0694] Wong et al., “Appearance of beta-lactamase activity in animalcells upon liposome-mediated gene transfer,” Gene., 10(2):87-94,1980.

[0695] Wu and Wu, “Receptor-mediated in vitro gene transfections by asoluble DNA carrier system,” J Biol. Chem., 262:4429-4432, 1987.

[0696] Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993.

[0697] Yagita, H., Nagata, M., Kawasaki, A., Shinkai, Y., and Okumura,K. Adv. Immunol. 1992; 51: 215-242.

[0698] Yang, X., Stennicke, H. R., Wang, B., Green, D. R., Janicke, R.U., Srinivasan, A., Seth, P., Salvesen, G., and Froelich, C. J. J. Biol.Chem. 1998; 273: 34278-34283.

[0699] Young J D-E, Hengartner H, Podack E R, Cohn Z A. Purification andcharacterization of a cytolytic pore-forming protein from granules ofcloned lymphocytes with natural killer activity. Cell 1986; 44: 849-859.

[0700] Young, J. D. E., and Cohn, Z. A. Cell. 1986; 46: 641-642.

[0701] Zamzami N., Susin S. A., Marchetti P., Hirsch T., Gomez-MonterreyI., Castedo M., and

[0702] Kroemer G. Mitochondrial control of nuclear apoptosis. J. Exp.Med., 183: 1533-1544, 1996.

[0703] Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ. BH3domain of BAD is required for heterodimerization with BCL-XL andpro-apoptotic activity. J Biol Chem. 1997 September 26;272(39):24101-4.

[0704] Zola, Monoclonal Antibodies: a Manual of Techniques, pp. 147-158(CRC Press, Inc., 1987). Zou H, Henzel W J, Liu X, Lutschg A, Wang X.Apaf-1, a human protein homologous to C. elegans CED-4, participates incytochrome c-dependent activation of caspase-3. Cell. 1997 August8;90(3):405-13.

[0705] Zunino S J, Bleackley R C, Martinez J, Hudig D. RNKP-1, a novelnatural killer cell-associated serine protease gene cloned from RNK-16cytotoxic lymphocytes. J Immunol 1990; 144: 2001-2009.

[0706] All of the methods and compositions disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe methods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents that are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1 61 1 36 DNA Artificial Sequence Description of Artificial SequencePrimer 1 ggtggcggtg gctccatgga accaatcctg cttctg 36 2 39 DNA ArtificialSequence Description of Artificial Sequence Primer 2 gccaccgcctccctcgagct attagtagcg tttcatggt 39 3 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 3 ggtaccgacg acgacgacaagatcatcggg ggacatgag 39 4 30 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 ggagccaccg ccaccgtagc gtttcatggt 30 5 30DNA Artificial Sequence Description of Artificial Sequence Primer 5ggtggcggtg gctccgcacc catggcagaa 30 6 42 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 aaggctcgtg tcgacctcgagtcattaccg cctcggcttg tc 42 7 48 DNA Artificial Sequence Description ofArtificial Sequence Primer 7 ggtggcggtg gctccacgga cattgtgatg acccagtctcaaaaattc 48 8 48 DNA Artificial Sequence Description of ArtificialSequence Primer 8 ggagccaccg ccaccctcga gctatcatga ggagacggtg agagtggt48 9 18 DNA Artificial Sequence Description of Artificial SequencePrimer 9 taatacgact cactatag 18 10 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 10 cttgtcgtcg tcgtcggtacccagatctgg 30 11 247 PRT Homo sapiens 11 Met Gln Pro Ile Leu Leu Leu LeuAla Phe Leu Leu Leu Pro Arg Ala 1 5 10 15 Asp Ala Gly Glu Ile Ile GlyGly His Glu Ala Lys Pro His Ser Arg 20 25 30 Pro Tyr Met Ala Tyr Leu MetIle Trp Asp Gln Lys Ser Leu Lys Arg 35 40 45 Cys Gly Gly Phe Leu Ile GlnAsp Asp Phe Val Leu Thr Ala Ala His 50 55 60 Cys Trp Gly Ser Ser Ile AsnVal Thr Leu Gly Ala His Asn Ile Lys 65 70 75 80 Glu Gln Glu Pro Thr GlnGln Phe Ile Pro Val Lys Arg Pro Ile Pro 85 90 95 His Pro Ala Tyr Asn ProLys Asn Phe Ser Asn Asp Ile Met Leu Leu 100 105 110 Gln Leu Glu Arg LysAla Lys Arg Thr Arg Ala Val Gln Pro Leu Arg 115 120 125 Leu Pro Ser AsnLys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val 130 135 140 Ala Gly TrpGly Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu 145 150 155 160 GlnGlu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp 165 170 175Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro 180 185190 Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val 195200 205 Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly210 215 220 Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His TrpIle 225 230 235 240 Lys Lys Thr Met Lys Arg Tyr 245 12 247 PRT Homosapiens 12 Met Gln Pro Ile Leu Leu Leu Leu Ala Phe Leu Leu Leu Pro ArgAla 1 5 10 15 Asp Ala Gly Glu Ile Ile Gly Gly His Glu Ala Lys Pro HisSer Arg 20 25 30 Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser LeuLys Arg 35 40 45 Cys Gly Gly Phe Leu Ile Arg Asp Asp Phe Val Leu Thr AlaAla His 50 55 60 Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His AsnIle Lys 65 70 75 80 Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys ArgPro Ile Pro 85 90 95 His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp IleMet Leu Leu 100 105 110 Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala ValGln Pro Leu Arg 115 120 125 Leu Pro Ser Asn Lys Ala Gln Val Lys Pro GlyGln Thr Cys Ser Val 130 135 140 Ala Gly Trp Gly Gln Thr Ala Pro Leu GlyLys His Ser His Thr Leu 145 150 155 160 Gln Glu Val Lys Met Thr Val GlnGlu Asp Arg Lys Cys Glu Ser Asp 165 170 175 Leu Arg His Tyr Tyr Asp SerThr Ile Glu Leu Cys Val Gly Asp Pro 180 185 190 Glu Ile Lys Lys Thr SerPhe Lys Gly Asp Ser Gly Gly Pro Leu Val 195 200 205 Cys Asn Lys Val AlaGln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly 210 215 220 Met Pro Pro ArgAla Cys Thr Lys Val Ser Ser Phe Val His Trp Ile 225 230 235 240 Lys LysThr Met Lys Arg Tyr 245 13 281 PRT Homo sapiens 13 Met Lys Ser Leu SerLeu Leu His Leu Phe Pro Leu Pro Arg Ala Lys 1 5 10 15 Arg Glu Gln GlyGly Asn Asn Ser Ser Ser Asn Gln Gly Ser Leu Pro 20 25 30 Glu Lys Met GlnPro Ile Leu Leu Leu Leu Ala Phe Leu Leu Leu Pro 35 40 45 Arg Ala Asp AlaGly Glu Ile Ile Gly Gly His Glu Ala Lys Pro His 50 55 60 Ser Arg Pro TyrMet Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu 65 70 75 80 Lys Arg CysGly Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr Ala 85 90 95 Ala His CysTrp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His Asn 100 105 110 Ile LysGlu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg Pro 115 120 125 IlePro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile Met 130 135 140Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln Pro 145 150155 160 Leu Arg Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr Cys165 170 175 Ser Val Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His SerHis 180 185 190 Thr Leu Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg LysCys Glu 195 200 205 Ser Asp Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu LeuCys Val Gly 210 215 220 Asp Pro Glu Ile Lys Lys Thr Ser Phe Lys Gly AspSer Gly Gly Pro 225 230 235 240 Leu Val Cys Asn Lys Val Ala Gln Gly IleVal Ser Tyr Gly Arg Asn 245 250 255 Asn Gly Met Pro Pro Arg Ala Cys ThrLys Val Ser Ser Phe Val His 260 265 270 Trp Ile Lys Lys Thr Met Lys ArgTyr 275 280 14 247 PRT Homo sapiens 14 Met Gln Pro Ile Leu Leu Leu LeuAla Phe Leu Leu Leu Pro Arg Ala 1 5 10 15 Asp Ala Gly Glu Ile Ile GlyGly His Glu Ala Lys Pro His Ser Arg 20 25 30 Pro Tyr Met Ala Tyr Leu MetIle Trp Asp Gln Lys Ser Leu Lys Arg 35 40 45 Cys Gly Gly Phe Leu Ile GlnAsp Asp Phe Val Leu Thr Ala Ala His 50 55 60 Cys Trp Gly Ser Ser Ile AsnVal Thr Leu Gly Ala His Asn Ile Lys 65 70 75 80 Glu Gln Glu Pro Thr GlnGln Phe Ile Pro Val Lys Arg Pro Ile Pro 85 90 95 His Pro Ala Tyr Asn ProLys Asn Phe Ser Asn Asp Ile Met Leu Leu 100 105 110 Gln Leu Glu Arg LysAla Lys Arg Thr Arg Ala Val Gln Pro Leu Arg 115 120 125 Leu Pro Ser AsnLys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val 130 135 140 Ala Gly TrpGly Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu 145 150 155 160 GlnGlu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp 165 170 175Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro 180 185190 Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val 195200 205 Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly210 215 220 Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His TrpIle 225 230 235 240 Lys Lys Thr Met Lys Arg Tyr 245 15 227 PRT Homosapiens 15 Ile Ile Gly Gly His Val Ala Lys Pro His Ser Arg Pro Tyr MetAla 1 5 10 15 Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg Cys GlyGly Phe 20 25 30 Leu Ile Arg Asp Asp Phe Val Leu Thr Ala Ala His Cys TrpGly Ser 35 40 45 Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys Glu GlnGlu Pro 50 55 60 Thr Gln Gln Phe Ile Pro Val Lys Arg Ala Ile Pro His ProAla Tyr 65 70 75 80 Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu GlnLeu Glu Arg 85 90 95 Lys Ala Lys Arg Thr Arg Ala Val Gln Pro Leu Arg LeuPro Ser Asn 100 105 110 Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser ValAla Gly Trp Gly 115 120 125 Gln Thr Ala Pro Leu Gly Lys His Ser His ThrLeu Gln Glu Val Lys 130 135 140 Met Thr Val Gln Glu Asp Arg Lys Cys GluSer Asp Leu Arg His Tyr 145 150 155 160 Tyr Asp Ser Thr Ile Glu Leu CysVal Gly Asp Pro Glu Ile Lys Lys 165 170 175 Thr Ser Phe Lys Gly Asp SerGly Gly Pro Leu Val Cys Asn Lys Val 180 185 190 Ala Gln Gly Ile Val SerTyr Gly Arg Asn Asn Gly Met Pro Pro Arg 195 200 205 Ala Cys Thr Lys ValSer Ser Phe Val His Trp Ile Lys Lys Thr Met 210 215 220 Lys Arg Tyr 22516 247 PRT Homo sapiens 16 Met Gln Pro Ile Leu Leu Leu Leu Ala Phe LeuLeu Leu Pro Arg Ala 1 5 10 15 Asp Ala Gly Glu Ile Ile Gly Gly His GluAla Lys Pro His Ser Arg 20 25 30 Pro Tyr Met Ala Tyr Leu Met Ile Trp AspGln Lys Ser Leu Lys Arg 35 40 45 Cys Gly Gly Phe Leu Ile Gln Asp Asp PheVal Leu Thr Ala Ala His 50 55 60 Cys Trp Gly Ser Ser Ile Asn Val Thr LeuGly Ala His Asn Ile Lys 65 70 75 80 Glu Gln Glu Pro Thr Gln Gln Phe IlePro Val Lys Arg Ala Ile Pro 85 90 95 His Pro Ala Tyr Asn Pro Lys Asn PheSer Asn Asp Ile Met Leu Leu 100 105 110 Gln Leu Glu Arg Lys Ala Lys ArgThr Arg Ala Val Gln Pro Leu Arg 115 120 125 Leu Pro Ser Asn Lys Ala GlnVal Lys Pro Gly Gln Thr Cys Ser Val 130 135 140 Ala Gly Trp Gly Gln ThrAla Pro Leu Gly Lys His Ser His Thr Leu 145 150 155 160 Gln Glu Val LysMet Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp 165 170 175 Leu Arg HisTyr Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro 180 185 190 Glu IleLys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val 195 200 205 CysAsn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly 210 215 220Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile 225 230235 240 Lys Lys Thr Met Lys Arg Tyr 245 17 889 DNA Homo sapiens 17gcagctccaa ccagggcagc cttcctgaga agatgcaacc aatcctgctt ctgctggcct 60tcctcctgct gcccagggca gatgcagggg agatcatcgg gggacatgag gccaagcccc 120actcccgccc ctacatggct tatcttatga tctgggatca gaagtctctg aagaggtgcg 180gtggcttcct gatacgagac gacttcgtgc tgacagctgc tcactgttgg ggaagctcca 240taaatgtcac cttgggggcc cacaatatca aagaacagga gccgacccag cagtttatcc 300ctgtgaaaag acccatcccc catccagcct ataatcctaa gaacttctcc aacgacatca 360tgctactgca gctggagaga aaggccaagc ggaccagagc tgtgcagccc ctcaggctac 420ctagcaacaa ggcccaggtg aagccagggc agacatgcag tgtggccggc tgggggcaga 480cggcccccct gggaaaacac tcacacacac tacaagaggt gaagatgaca gtgcaggaag 540atcgaaagtg cgaatctgac ttacgccatt attacgacag taccattgag ttgtgcgtgg 600gggacccaga gattaaaaag acttccttta agggggactc tggaggccct cttgtgtgta 660acaaggtggc ccagggcatt gtctcctatg gacgaaacaa tggcatgcct ccacgagcct 720gcaccaaagt ctcaagcttt gtacactgga taaagaaaac catgaaacgc tactaactac 780aggaagcaaa ctaagccccc gctgtaatga aacaccttct ctggagccaa gtccagattt 840acactgggag aggtgccagc aactgaataa atacctctta gctgagtgg 889 18 475 DNAHomo sapiens 18 ttccactcag ctaagaggta tttattcagt tgctggcacc tctcccagtgtaaatctgga 60 cttggctcca gagaaggtgt ttcattacag cgggggctta gtttgcttcctgtagttagt 120 agcgtttcat ggttttcttt atccagtgta caaagcttga gactttggtgcaggctcgtg 180 gaggcatgcc attgtttcgt ccataggaga caatgccctg ggccaccttgttacacacaa 240 gagggcctcc agagtccccc ttaaaggaag tctttttaat ctctgggtcccccacgcaca 300 actcaatggt actgtcgtaa taatggcgta agtcagattc gcactttcgatcttcctgca 360 ctgtcatctt cacctcttgt agtgtgtgtg agtgttttcc caagggggccgtctgccccc 420 aaccggccac actggatgtt tgccctggct tttacctggc cttgttgctatgtaa 475 19 484 DNA Homo sapiens modified_base (438) N = A, C, G or T/U19 tttttccact cagctaagag gtatttattc agttgctggc accctctccc agtgtaaatc 60tggacttggc tccagagaag gtgtttcatt acagcggggg cttagtttgc ttcctgtagt 120tagtagcgtt tcatggtttt ctttatccag tgtacaaagc ttgagacttt ggtgcaggct 180cgtggaggca tgccattgtt tcgtccatag gagacaatgc cctgggccac cttgttacac 240acaagagggc ctccagagtc ccccttaaag gaagtctttt taatctctgg gtcccccacg 300cacaactcaa tggtactgtc gtaataatgg cgtaagtcag attcgcactt tcgatcttcc 360tgcactgtca tcttcacctc ttgtagtgtg tgtgagtgtt ttcccagggg ggccgtttgc 420cccaaccggc cacactgnat gtttgtcctt ggttcacctg ggcccttggt gctaggtagc 480cccg 484 20 960 DNA Homo sapiens 20 agcagctcca accagggcag ccttcctgagaagatgcaac caatcctgct tctgctggcc 60 ttcctcctgc tgcccagggc agatgcaggggagatcatcg ggggacatga ggccaagccc 120 cactcccgcc cctacatggc ttatcttatgatctgggatc agaagtctct gaagaggtgc 180 ggtggcttcc tgatacaaga cgacttcgtgctgacagctg ctcactgttg gggaagctcc 240 ataaatgtca ccttgggggc ccacaatatcaaagaacagg agccgaccca gcagtttatc 300 cctgtgaaaa gacccatccc ccatccagcctataatccta agaacttctc caacgacatc 360 atgctactgc agctggagag aaaggccaagcggaccagag ctgtgcagcc cctcaggcta 420 cctagcaaca aggcccaggt gaagccagggcagacatgca gtgtggccgg ctgggggcag 480 acggcccccc tgggaaaaca ctcacacacactacaagagg tgaagatgac agtgcaggaa 540 gatcgaaagt gcgaatctga cttacgccattattacgaca gtaccattga gttgtgcgtg 600 ggggacccag agattaaaaa gacttcctttaagggggact ctggaggccc tcttgtgtgt 660 aacaaggtgg cccagggcat tgtctcctatggacgaaaca atggcatgcc tccacgagcc 720 tgcaccaaag tctcaagctt tgtacactggataaagaaaa ccatgaaacg ctactaacta 780 caggaagcaa actaagcccc cgctgtaatgaaacaccttc tctggagcca agtccagatt 840 tacactggga gaggtgccag caactgaataaatacctctc ccagtgtaaa tctggagcca 900 agtccagatt tacactggga gaggtgccagcaactgaata aatacctctt agctgagtgg 960 21 831 DNA Homo sapiens 21atcatcgggg gacatgtggc caagccccac tcccgcccct acatggctta tcttatgatc 60tgggatcaga agtctctgaa gaggtgcggt ggcttcctga tacgagacga cttcgtgctg 120acagctgctc actgttgggg aagctccata aatgtcacct tgggggccca caatatcaag 180gaacaggagc cgacccagca gtttatccct gtgaaaagag ccatccccca tccagcctat 240aatcctaaga acttctccaa tgacatcatg ctactgcagc tggagagaaa ggccaagcgg 300accagagctg tgcagcccct caggctacct agcaacaagg cccaggtgaa gccagggcag 360acatgcagtg tggccggctg ggggcagacg gcccccctgg gaaaacattc acacacacta 420caagaggtga agatgacagt gcaggaagat cgaaagtgcg aatctgactt acgccattat 480tacgacagta ccattgagtt gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag 540ggggactctg gaggccctct tgtgtgtaac aaggtggccc agggcattgt ctcctatgga 600cgaaacaatg gcatgcctcc acgagcctgc accaaagtct caagctttgt acactggata 660aagaaaacca tgaaacgcta ctaactacag gaagcaaact aagcccccgc tgtaatgcta 720caggaagcaa actaagcccc cgctgtaatg aaacaccttc tctggagcca agtccagatt 780tacactggga gaggtgccag caactgaata aatacctctt agctgagtgg t 831 22 4751 DNAHomo sapiens 22 gaattctata ttttgagata taccattcct catagaaaaa tttcctcacagaaaatataa 60 aggtggaaac aaatcacaag aatcgaacca tgtagagaga cttagttgtcttttaacaga 120 attgggcacg ggctgttcag aaacaacaat ctttcacatc cattataatgatagcattag 180 tgtagtttgt ttagcaaatg tttactgcga gcctgttatg tgctgagcctgctatgtaag 240 aagtgtggct ctctggacag gagacagaat actaaacaac acaactactgatctttggct 300 gcctggcatg cttcctcact tcatatggta tcagcaattt agcaccacaaacgtccttta 360 gagaccagcc ctttctcatt cttggttcta gtggcttgag tagactgaccccactaccca 420 agtggatttg actcctagca attcattaat ctagcccata aatgtcaagtacaggacttt 480 attgaagcat tcagaaagag gaatagggga tgttagaatc tctagaaaggaagctatgat 540 aataaatggg ttgctagatg ggtctagtag atggtggcca tgctttgttactgccttgtg 600 tattgtgcta ccatagccct ccccaaactg tactctggct cctggcatttccgtctcttc 660 aaccagatgg tcagctctct aagtgaagga gacacatctc caacatgcttggttctagca 720 caacagaagg gctcaaacac atacctgcta aagaaactat cctgatggatttagcagcat 780 ggccatgagg cattggcggt tctatcactg ggaactcagg tttctggtgctccagtacct 840 ctactggctg ataccacatc ctacagttca cttcataggc ttgggttcctgctctgggct 900 gaataggtgg tccactctga gtcatcagct gtggtgatga tgtggtcactgcatgattct 960 cacacaagca cccagaggac gtcatcaggc agaggcagtg ggggtgggcagcatttacag 1020 aaaatctgtg atgagacacc acaaaaccag aggggaacat gaagtcactgagcctgctcc 1080 acctctttcc tctcccaaga gctaaaagag agcaaggagg aaacaacagcagctccaacc 1140 agggcagcct tcctgagaag atgcaaccaa tcctgcttct gctggccttcctcctgctgc 1200 ccagggcaga tgcaggtgag tgaccgtctt ccaacctcgg ggcccaacccatcccacagg 1260 tctcctgccc tttctccaca ttcctgatcc atctatctac caggaatgttctgaactcca 1320 gctcccattc taccaagacc ccccaagtgt gatgctggat aagctatcagcaggaatggc 1380 agagcagcag gccattctca agaagagcca gtgggtacta tcccttccccagagcccacc 1440 tttgtcacct ggagagtagg actttcctag aagtaaatgg cagaggatgggaaactagaa 1500 aagagaaata ttaaattatt ctagagtagg cctggcttct gtttctgggataagacaggt 1560 gcttctctca ctgtacttag gagagaaacc cagagctcag ctgacagcagaattggtaca 1620 atcactgtcc tcagaacact gttaatgtgt ttgctcagtc ccattctccaactctgcttt 1680 tcttccctgg cctttggtgg ctcccctctt tccaaggatg aggcactacggcaggcccca 1740 gcttccctgc tttctagaat tccaccagca ctgctctacc agccctcatccagaggctaa 1800 ctggagccag tccatcatgc agccatgaac atttactggg cacccactacatgtcaggct 1860 ctaggaaaca ggatatgaca gtatctagat ccctccactt acaccctggccattagaaag 1920 cagcactatc ctagacacca caggactcat aagggtcttg gaaactcacctgaaacaaag 1980 caaagtcagg agaggaatga tcaggagcct ctgggatttc actgtccctaagacaggtat 2040 gctcgccttc aactacatat ggaagaaaga tttacagacc aaagtctgctgttcttccct 2100 ttttcagagc aggaaattga agccccttcc tccaggccac tcccaactccaggctatccc 2160 aggctcccaa atgcccagga gttctggagc cactaagcag gtgcccacccagcagattcc 2220 atgggtgccc acaagcagac agacttttcc ttcaggggag atcatcgggggacatgaggc 2280 caagccccac tcccgcccct acatggctta tcttatgatc tgggatcagaagtctctgaa 2340 gaggtgcggt ggcttcctga tacaagacga cttcgtgctg acagctgctcactgttgggg 2400 aaggtgagga gcagaaaaca gcccacaccc tcctggaaac actccacagagacccctgcc 2460 ttcttcccaa ggagctccct gggctcctgt gaacacacat gccaggaggtctccttagag 2520 ggtgagaaaa gggcagttaa gtttgtggag agaggggaag gttggttccagaggtgctgc 2580 tgaagtaaga aacagcagag tgaccaagcc tgccatattt agaactgggggcatactttg 2640 gcatagaata caaactgaag caattccacc tgtgtttcta gggggaaccgaaccctgaga 2700 aacctggtgc aattaccaga attccaattc ctggggaccg actgtccttaatttcccctc 2760 agctgcagcc ctgccccagc tgtcacctgc tcttcactgt ctctgggctgtatacactgt 2820 gactccaccc ccatcctcac tctgctctct gtgcagctcc ataaatgtcaccttgggggc 2880 ccacaatatc aaggaacagg agccgaccca gcagtttatc cctgtgaaaagagccatccc 2940 ccatccagcc tataatccta agaacttctc caatgacatc atgctactgcaggtgaggca 3000 cactcctgcc actcttgctc ttcttggtcc agttggttcc actccccctggaatgccggc 3060 ccttccctcc tttccatcct ggcctcttgg ttagttccta tgcctcagaggagagaggga 3120 agattgtgca gccccatcac tgtgtcgggg cccagaagtt cgttggctgacctggacttt 3180 cttgcctctt ccccaccagc tggagagaaa ggccaagcgg accagagctgtgcagcccct 3240 caggctacct agcaacaagg cccaggtgaa gccagggcag acatgcagtgtggccggctg 3300 ggggcagacg gcccccctgg gaaaacactc acacacacta caagaggtgaagatgacagt 3360 gcaggaagat cgaaagtgcg aatctgactt acgccattat tacgacagtaccattgagtt 3420 gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag gtaagactatgcacctgcct 3480 ggattggctc ttgggagaaa gatgtttggg gaatatctga gacctggagactcaagtagt 3540 gggggactcc ttcacccact agactgtgat atttctctct ggaaagagaaaaggggacta 3600 gactgagctg gggagaaatt agggcctctg caaacttacc aagaggccttatggtggatg 3660 gtgccttctt tggaaggatg aatttgcaac actccaccca ctccaggtcacagatattag 3720 gaaactgtgc ccatgggggt gcagctaatt ataaccaggt gtgtcttcagaggctggtac 3780 ccaacgtggt taatgggctg gtcctccatg gtggacatca gccctccttgcccacttctg 3840 ggtccttaaa cagccaacgg tcccacatac ctccgatctc aggatctgggggacatgacg 3900 gaggctggcc cctgggatga ggtgaagcag taacaatgtc cagggccagagcttggcagc 3960 tggggccacc agcggcctgc cctgccctct ggtctcccac atgtaggctgtgcaagttgg 4020 ccttttctaa aagggggctt gagatggaag agagggcagg acccggaggagcatcagctc 4080 agtccttcca ctctctattc acagggggac tctggaggcc ctcttgtgtgtaacaaggtg 4140 gcccagggca ttgtctccta tggacgaaac aatggcatgc ctccacgagcctgcaccaaa 4200 gtctcaagct ttgtacactg gataaagaaa accatgaaac gctactaactacaggaagca 4260 aactaagccc ccgctgtaat gaaacacctt ctctggagcc aagtccagatttacactggg 4320 agaggtgcca gcaactgaat aaatacctct tagctgagtg gaaagctggtttcttgttta 4380 ttcattgacc ctcattctca ggcaccacat ctgcgctatg caggccaatgacacaatttt 4440 gctgttttct gctttctcct ctcccctcac cccttgccac ctccccaaacccccacatga 4500 agctgatact cagctccttc ctatccacac cagtttctcc agggcctgccttctgccaag 4560 gctgaagctg agcaccatca ggagacaaca tggaccactt tggtcctggggctttgggta 4620 aacttcttac ctccttctcc agtgttacat tgacagagaa aaaagggataataccatggg 4680 acctaactcc tcatcccact ggggctcctc attctcccct gggcttagtttctctaccct 4740 cctctgagct c 4751 23 262 PRT Homo sapiens 23 Met Arg AsnSer Tyr Arg Phe Leu Ala Ser Ser Leu Ser Val Val Val 1 5 10 15 Ser LeuLeu Leu Ile Pro Glu Asp Val Cys Glu Lys Ile Ile Gly Gly 20 25 30 Asn GluVal Thr Pro His Ser Arg Pro Tyr Met Val Leu Leu Ser Leu 35 40 45 Asp ArgLys Thr Ile Cys Ala Gly Ala Leu Ile Ala Lys Asp Trp Val 50 55 60 Leu ThrAla Ala His Cys Asn Leu Asn Lys Arg Ser Gln Val Ile Leu 65 70 75 80 GlyAla His Ser Ile Thr Arg Glu Glu Pro Thr Lys Gln Ile Met Leu 85 90 95 ValLys Lys Glu Phe Pro Tyr Pro Cys Tyr Asp Pro Ala Thr Arg Glu 100 105 110Gly Asp Leu Lys Leu Leu Gln Leu Thr Glu Lys Ala Lys Ile Asn Lys 115 120125 Tyr Val Thr Ile Leu His Leu Pro Lys Lys Gly Asp Asp Val Lys Pro 130135 140 Gly Thr Met Cys Gln Val Ala Gly Trp Gly Arg Thr His Asn Ser Ala145 150 155 160 Ser Trp Ser Asp Thr Leu Arg Glu Val Asn Ile Thr Ile IleAsp Arg 165 170 175 Lys Val Cys Asn Asp Arg Asn His Tyr Asn Phe Asn ProVal Ile Gly 180 185 190 Met Asn Met Val Cys Ala Gly Ser Leu Arg Gly GlyArg Asp Ser Cys 195 200 205 Asn Gly Asp Ser Gly Ser Pro Leu Leu Cys GluGly Val Phe Arg Gly 210 215 220 Val Thr Ser Phe Gly Leu Glu Asn Lys CysGly Asp Pro Arg Gly Pro 225 230 235 240 Gly Val Tyr Ile Leu Leu Ser LysLys His Leu Asn Trp Ile Ile Met 245 250 255 Thr Ile Lys Gly Ala Val 26024 262 PRT Homo sapiens 24 Met Arg Asn Ser Tyr Arg Phe Leu Ala Ser SerLeu Ser Val Val Val 1 5 10 15 Ser Leu Leu Leu Ile Pro Glu Asp Val CysGlu Lys Ile Ile Gly Gly 20 25 30 Asn Glu Val Thr Pro His Ser Arg Pro TyrMet Val Leu Leu Ser Leu 35 40 45 Asp Arg Lys Thr Ile Cys Ala Gly Ala LeuIle Ala Lys Asp Trp Val 50 55 60 Leu Thr Ala Ala His Cys Asn Leu Asn LysArg Ser Gln Val Ile Leu 65 70 75 80 Gly Ala His Ser Ile Thr Arg Glu GluPro Thr Lys Gln Ile Met Leu 85 90 95 Val Lys Lys Glu Phe Pro Tyr Pro CysTyr Asp Pro Ala Thr Arg Glu 100 105 110 Gly Asp Leu Lys Leu Leu Gln LeuMet Glu Lys Ala Lys Ile Asn Lys 115 120 125 Tyr Val Thr Ile Leu His LeuPro Lys Lys Gly Asp Asp Val Lys Pro 130 135 140 Gly Thr Met Cys Gln ValAla Gly Trp Gly Arg Thr His Asn Ser Ala 145 150 155 160 Ser Trp Ser AspThr Leu Arg Glu Val Asn Ile Thr Ile Ile Asp Arg 165 170 175 Lys Val CysAsn Asp Arg Asn His Tyr Asn Phe Asn Pro Val Ile Gly 180 185 190 Met AsnMet Val Cys Ala Gly Ser Leu Arg Gly Gly Arg Asp Ser Cys 195 200 205 AsnGly Asp Ser Gly Ser Pro Leu Leu Cys Glu Gly Val Phe Arg Gly 210 215 220Val Thr Ser Phe Gly Leu Glu Asn Lys Cys Gly Asp Pro Arg Gly Pro 225 230235 240 Gly Val Tyr Ile Leu Leu Ser Lys Lys His Leu Asn Trp Ile Ile Met245 250 255 Thr Ile Lys Gly Ala Val 260 25 262 PRT Homo sapiens MOD_RES(121) x = t or m 25 Met Arg Asn Ser Tyr Arg Phe Leu Ala Ser Ser Leu SerVal Val Val 1 5 10 15 Ser Leu Leu Leu Ile Pro Glu Asp Val Cys Glu LysIle Ile Gly Gly 20 25 30 Asn Glu Val Thr Pro His Ser Arg Pro Tyr Met ValLeu Leu Ser Leu 35 40 45 Asp Arg Lys Thr Ile Cys Ala Gly Ala Leu Ile AlaLys Asp Trp Val 50 55 60 Leu Thr Ala Ala His Cys Asn Leu Asn Lys Arg SerGln Val Ile Leu 65 70 75 80 Gly Ala His Ser Ile Thr Arg Glu Glu Pro ThrLys Gln Ile Met Leu 85 90 95 Val Lys Lys Glu Phe Pro Tyr Pro Cys Tyr AspPro Ala Thr Arg Glu 100 105 110 Gly Asp Leu Lys Leu Leu Gln Leu Xaa GluLys Ala Lys Ile Asn Lys 115 120 125 Tyr Val Thr Ile Leu His Leu Pro LysLys Gly Asp Asp Val Lys Pro 130 135 140 Gly Thr Met Cys Gln Val Ala GlyTrp Gly Arg Thr His Asn Ser Ala 145 150 155 160 Ser Trp Ser Asp Thr LeuArg Glu Val Asn Ile Thr Ile Ile Asp Arg 165 170 175 Lys Val Cys Asn AspArg Asn His Tyr Asn Phe Asn Pro Val Ile Gly 180 185 190 Met Asn Met ValCys Ala Gly Ser Leu Arg Gly Gly Arg Asp Ser Cys 195 200 205 Asn Gly AspSer Gly Ser Pro Leu Leu Cys Glu Gly Val Phe Arg Gly 210 215 220 Val ThrSer Phe Gly Leu Glu Asn Lys Cys Gly Asp Pro Arg Gly Pro 225 230 235 240Gly Val Tyr Ile Leu Leu Ser Lys Lys His Leu Asn Trp Ile Ile Met 245 250255 Thr Ile Lys Gly Ala Val 260 26 878 DNA Homo sapiens 26 cagattttcaggttgattga tgtgggacag cagccacaat gaggaactcc tatagatttc 60 tggcatcctctctctcagtt gtcgtttctc tcctgctaat tcctgaagat gtctgtgaaa 120 aaattattggaggaaatgaa gtaactcctc attcaagacc ctacatggtc ctacttagtc 180 ttgacagaaaaaccatctgt gctggggctt tgattgcaaa agactgggtg ttgactgcag 240 ctcactgtaacttgaacaaa aggtcccagg tcattcttgg ggctcactca ataaccaggg 300 aagagccaacaaaacagata atgcttgtta agaaagagtt tccctatcca tgctatgacc 360 cagccacacgcgaaggtgac cttaaacttt tacagctgat ggaaaaagca aaaattaaca 420 aatatgtgactatccttcat ctacctaaaa agggggacga tgtgaaacca ggaaccatgt 480 gccaagttgcagggtggggc aggactcaca atagtgcatc ttggtccgat actctgagag 540 aagtcaatatcaccatcata gacagaaaag tctgcaatga tcgaaatcac tataatttta 600 accctgtgattggaatgaat atggtttgtg ctggaagcct ccgaggtgga agagactcgt 660 gcaatggagattctggaagc cctttgttgt gcgagggtgt tttccgaggg gtcacttcct 720 ttggccttgaaaataaatgc ggagaccctc gtgggcctgg tgtctatatt cttctctcaa 780 agaaacacctcaactggata attatgacta tcaagggagc agtttaaata accgtttcct 840 ttcatttactgtggcttctt aatcttttca caaataaa 878 27 878 DNA Homo sapiens 27 cagattttcaggttgattga tgtgggacag cagccacaat gaggaactcc tatagatttc 60 tggcatcctctctctcagtt gtcgtttctc tcctgctaat tcctgaagat gtctgtgaaa 120 aaattattggaggaaatgaa gtaactcctc attcaagacc ctacatggtc ctacttagtc 180 ttgacagaaaaaccatctgt gctggggctt tgattgcaaa agactgggtg ttgactgcag 240 ctcactgtaacttgaacaaa aggtcccagg tcattcttgg ggctcactca ataaccaggg 300 aagagccaacaaaacagata atgcttgtta agaaagagtt tccctatcca tgctatgacc 360 cagccacacgcgaaggtgac cttaaacttt tacagctgac ggaaaaagca aaaattaaca 420 aatatgtgactatccttcat ctacctaaaa agggggatga tgtgaaacca ggaaccatgt 480 gccaagttgcagggtggggg aggactcaca atagtgcatc ttggtccgat actctgagag 540 aagtcaatatcaccatcata gacagaaaag tctgcaatga tcgaaatcac tataatttta 600 accctgtgattggaatgaat atggtttgtg ctggaagcct ccgaggtgga agagactcgt 660 gcaatggagattctggaagc cctttgttgt gcgagggtgt tttccgaggg gtcacttcct 720 ttggccttgaaaataaatgc ggagaccctc gtgggcctgg tgtctatatt cttctctcaa 780 agaaacacctcaactggata attatgacta tcaagggagc agtttaaata accgtttcct 840 ttcatttactgtggcttctt aatcttttca caaataaa 878 28 1610 DNA Homo sapiens 28aagcttccaa tgactttctt cacagaattg gaaaaaacta ctttaaagtt catatggaac 60caaacaagag cccacattgc caagacaatc ctaagccaaa agaacaaagc tggaagcatc 120atgctacctg acttcaacaa tactacaagg ctacgtaacc aaaacagatg gactggtaat 180ggctgcacaa ctatgcatat atactaaatc cattgactat acactctaaa tgggtgacct 240tatggtgtgt gaattatgtc tcataaagtt gttagaagtc gacataaatg gaagagcaac 300cattcacata aaaataaaca aaattgtcaa tgttttaaga atttttcagt aggtgtagtt 360aattacaatt tgactttttt aagtctgcac taaattactc accaaaacca atagcagggt 420cctcactgct gttactgaaa atgattaacc tttgatacac ttgtaatatc tgagaaaaag 480aaatgcaggg gtctcagcag ggctcccttc taaggtcact tgatttctaa agaagtaacc 540actaggtttg aagtcatcag gatgttaact atggggatgg ttggttcagt acccaacatc 600ctgacagcac atctgaccat gtatattgta tcggagacca catcctcagc tcagaaaaag 660agctgaactc atttcaaata gaagcacacc tgcatactgt tcctccaggg actgaggttg 720actcttctta gagtgagaca ttccccaaca ttggaacaaa aatgactccc acttcttttc 780tcacctaaac ctgttcagaa gaaagaagaa aggcaggaag caggggtcgg ggggggcggg 840gagggaggga aactcggaga tactttcagt atctaaagtt gtgaaactag acaatcagga 900acgcacaatc agagggctga aaagggccaa gagcccccta ccctcctcca gcccatgttc 960ccacacctgc cacagaccag gcaggagcag aataaacact cacacaaagt gggaaggaaa 1020atccagcagg agcctctatg taaataaatc tccctcctgt cctgagcttg cacttggcct 1080gctaattcta tataacccaa ggagacagct agaaagaatt ttgattggtg accaattttg 1140aggactttta ttaaaattct aatttaagtc ttcgagagtt tccagtcatg gatagtacag 1200ataatattgc agatgatgaa agcgtcttca aaatcatagc tgagaccttc acgtcttccc 1260tggtgtactc tgatggcaac aaggtccctt gcccctctcc tacccatgta gaattccagc 1320gcccccctca gcagtcctag caaaggaaag cctgcctgct ggcagtgagc catcatccac 1380cattctcact tatttggatt tggtttccta atgctaaact ttgaaacttg aaaaaaaaaa 1440tgaaaggaaa ccacatcctt tttactctca gtatatagat agaggcagtt aagaactgaa 1500aacagatttt caggttgatt gatgtgggac agcagccaca atgaggaact cctatagatt 1560tctggcatcc tctctctcag ttgtcgtttc tctcctgcta attcctgaag 1610 29 24 DNAArtificial Sequence Description of Artificial Sequence Primer 29ggtgatggac gggtccgggg agca 24 30 30 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 30 ggcctcagcc catcttcttc cagatggtga 30 3151 DNA Artificial Sequence Description of Artificial Sequence Primer 31ggtggcggtg gctccatggc ggacattgtg atgacccagt ctcaaaaatt c 51 32 43 DNAArtificial Sequence Description of Artificial Sequence Primer 32cgtcggagcc accgccaccg ctagctgagg agacggtgag agt 43 33 33 DNA ArtificialSequence Description of Artificial Sequence Primer 33 ggtggcggtggctccgacgg gtccggggag cag 33 34 45 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 34 ggagccaccg ccaccctcga gctatcagcccatcttcttc cagat 45 35 36 DNA Artificial Sequence Description ofArtificial Sequence Primer 35 ggtggcggtg gctccatgga cgggtccggg gagcag 3636 42 DNA Artificial Sequence Description of Artificial Sequence Primer36 gtccgtggag ccaccgccac cgctagcgcc catcttcttc ca 42 37 48 DNAArtificial Sequence Description of Artificial Sequence Primer 37ggtggcggtg gctccacgga cattgtgatg acccagtctc aaaaattc 48 38 48 DNAArtificial Sequence Description of Artificial Sequence Primer 38ggagccaccg ccaccctcga gctatcatga ggagacggtg agagtggt 48 39 42 DNAArtificial Sequence Description of Artificial Sequence Primer 39ggagccaccg ccaccctcga gctatcacca accaccctgg tc 42 40 30 DNA ArtificialSequence Description of Artificial Sequence Primer 40 ggagccaccgccaccccaac caccctggtc 30 41 37 DNA Artificial Sequence Description ofArtificial Sequence Primer 41 ccggagccac cgccaccgct agctgaggag actgtga37 42 31 DNA Artificial Sequence Description of Artificial SequencePrimer 42 ggtggcggtg gctccttcat ccaggatcga g 31 43 34 DNA ArtificialSequence Description of Artificial Sequence Primer 43 ggtggcggtggctccatggt catccaggat cgag 34 44 57 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 44 aaacatgcca tggctcacca ccaccaccaccacgacgggt ccggggagca gcccaga 57 45 573 DNA Homo sapiens 45 gacgggtccggggagcagcc cagaggcggg gggcccacca gctctgagca gatcatgaag 60 acaggggcccttttgcttca gggtttcatc caggatcgag cagggcgaat ggggggggag 120 gcacccgagctggccctgga cccggtgcct caggatgcgt ccaccaagaa gctgagcgag 180 tgtctcaagcgcatcgggga cgaactggac agtaacatgg agctgcagag gatgattgcc 240 gccgtggacacagactcccc ccgagaggtc tttttccgag tggcagctga catgttttct 300 gacggcaacttcaactgggg ccgggttgtc gcccttttct actttgccag caaactggtg 360 ctcaaggccctgtgcaccaa ggtgccggaa ctgatcagaa ccatcatggg ctggacattg 420 gacttcctccgggagcggct gttgggctgg atccaagacc agggtggttg ggacggcctc 480 ctctcctactttgggacgcc cacgtggcag accgtgacca tctttgtggc gggagtgctc 540 accgcctcgctcaccatctg gaagaagatg ggc 573 46 191 PRT Homo sapiens 46 Asp Gly Ser GlyGlu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser Glu 1 5 10 15 Gln Ile MetLys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln Asp 20 25 30 Arg Ala GlyArg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp Pro 35 40 45 Val Pro GlnAsp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys Arg 50 55 60 Ile Gly AspGlu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile Ala 65 70 75 80 Ala ValAsp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala Ala 85 90 95 Asp MetPhe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala Leu 100 105 110 PheTyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Val Thr Lys Val 115 120 125Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu Arg 130 135140 Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly Leu 145150 155 160 Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gln Thr Val Thr Ile PheVal 165 170 175 Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys MetGly 180 185 190 47 11 PRT Homo sapiens 47 Asp Gly Asn Phe Asn Trp GlyArg Val Val Ala 1 5 10 48 9 PRT Homo sapiens 48 Trp Ile Gln Asp Gln GlyGly Trp Asp 1 5 49 7 PRT Homo sapiens 49 Leu Lys Arg Ile Gly Asp Glu 1 550 5 PRT Artificial Sequence Description of Artificial Sequence Linker50 Gly Gly Gly Gly Ser 1 5 51 14 PRT Artificial Sequence Description ofArtificial Sequence Linker 51 Glu Gly Lys Ser Ser Gly Ser Gly Ser GluSer Lys Val Asp 1 5 10 52 18 PRT Artificial Sequence Description ofArtificial Sequence Linker 52 Lys Glu Ser Gly Ser Val Ser Ser Glu GlnLeu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp 53 4 PRT Artificial SequenceDescription of Artificial Sequence Enterokinase cleavage site 53 Asp AspAsp Lys 1 54 744 DNA Homo sapiens 54 atgcaaccaa tcctgcttct gctggccttcctcctgctgc ccagggcaga tgcaggggag 60 atcatcgggg gacatgaggc caagccccactcccgcccct acatggctta tcttatgatc 120 tgggatcaga agtctctgaa gaggtgcggtggcttcctga tacaagacga cttcgtgctg 180 acagctgctc actgttgggg aagctccataaatgtcacct tgggggccca caatatcaaa 240 gaacaggagc cgacccagca gtttatccctgtgaaaagac ccatccccca tccagcctat 300 aatcctaaga acttctccaa cgacatcatgctactgcagc tggagagaaa ggccaagcgg 360 accagagctg tgcagcccct caggctacctagcaacaagg cccaggtgaa gccagggcag 420 acatgcagtg tggccggctg ggggcagacggcccccctgg gaaaacactc acacacacta 480 caagaggtga agatgacagt gcaggaagatcgaaagtgcg aatctgactt acgccattat 540 tacgacagta ccattgagtt gtgcgtgggggacccagaga ttaaaaagac ttcctttaag 600 ggggactctg gaggccctct tgtgtgtaacaaggtggccc agggcattgt ctcctatgga 660 cgaaacaatg gcatgcctcc acgagcctgcaccaaagtct caagctttgt acactggata 720 aagaaaacca tgaaacgcta ctaa 744 55247 PRT Homo sapiens 55 Met Gln Pro Ile Leu Leu Leu Leu Ala Phe Leu LeuLeu Pro Arg Ala 1 5 10 15 Asp Ala Gly Glu Ile Ile Gly Gly His Glu AlaLys Pro His Ser Arg 20 25 30 Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp GlnLys Ser Leu Lys Arg 35 40 45 Cys Gly Gly Phe Leu Ile Gln Asp Asp Phe ValLeu Thr Ala Ala His 50 55 60 Cys Trp Gly Ser Ser Ile Asn Val Thr Leu GlyAla His Asn Ile Lys 65 70 75 80 Glu Gln Glu Pro Thr Gln Gln Phe Ile ProVal Lys Arg Pro Ile Pro 85 90 95 His Pro Ala Tyr Asn Pro Lys Asn Phe SerAsn Asp Ile Met Leu Leu 100 105 110 Gln Leu Glu Arg Lys Ala Lys Arg ThrArg Ala Val Gln Pro Leu Arg 115 120 125 Leu Pro Ser Asn Lys Ala Gln ValLys Pro Gly Gln Thr Cys Ser Val 130 135 140 Ala Gly Trp Gly Gln Thr AlaPro Leu Gly Lys His Ser His Thr Leu 145 150 155 160 Gln Glu Val Lys MetThr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp 165 170 175 Leu Arg His TyrTyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro 180 185 190 Glu Ile LysLys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val 195 200 205 Cys AsnLys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly 210 215 220 MetPro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile 225 230 235240 Lys Lys Thr Met Lys Arg Tyr 245 56 1059 DNA Artificial SequenceDescription of Artificial Sequence Granhzyme B-vegf121 56 atcatcgggggacatgaggc caagccccac tcccgcccct acatggctta tcttatgatc 60 tgggatcagaagtctctgaa gaggtgcggt ggcttcctga tacaagacga cttcgtgctg 120 acagctgctcactgttgggg aagctccata aatgtcacct tgggggccca caatatcaaa 180 gaacaggagccgacccagca gtttatccct gtgaaaagac ccatccccca tccagcctat 240 aatcctaagaacttctccaa cgacatcatg ctactgcagc tggagagaaa ggccaagcgg 300 accagagctgtgcagcccct caggctacct agcaacaagg cccaggtgaa gccagggcag 360 acatgcagtgtggccggctg ggggcagacg gcccccctgg gaaaacactc acacacacta 420 caagaggtgaagatgacagt gcaggaagat cgaaagtgcg aatctgactt acgccattat 480 tacgacagtaccattgagtt gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag 540 ggggactctggaggccctct tgtgtgtaac aaggtggccc agggcattgt ctcctatgga 600 cgaaacaatggcatgcctcc acgagcctgc accaaagtct caagctttgt acactggata 660 aagaaaaccatgaaacgcta cggtggcggt ggctccgcac ccatggcaga aggaggaggg 720 cagaatcatcacgaagtggt gaagttcatg gatgtctatc agcgcagcta ctgccatcca 780 atcgagaccctggtggacat cttccaggag taccctgatg agatcgagta catcttcaag 840 ccatcctgtgtgcccctgat gcgatgcggg ggctgctgca atgacgaggg cctggagtgt 900 gtgcccactgaggagtccaa catcaccatg cagattatgc ggatcaaacc tcaccaaggc 960 cagcacataggagagatgag cttcctacag cacaacaaat gtgaatgcag accaaagaaa 1020 gatagagcaagacaagaaaa ttgtgacaag ccgaggcgg 1059 57 353 PRT Artificial SequenceDescription of Artificial Sequence Granzyme B-vegf121 57 Ile Ile Gly GlyHis Glu Ala Lys Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 Tyr Leu MetIle Trp Asp Gln Lys Ser Leu Lys Arg Cys Gly Gly Phe 20 25 30 Leu Ile GlnAsp Asp Phe Val Leu Thr Ala Ala His Cys Trp Gly Ser 35 40 45 Ser Ile AsnVal Thr Leu Gly Ala His Asn Ile Lys Glu Gln Glu Pro 50 55 60 Thr Gln GlnPhe Ile Pro Val Lys Arg Pro Ile Pro His Pro Ala Tyr 65 70 75 80 Asn ProLys Asn Phe Ser Asn Asp Ile Met Leu Leu Gln Leu Glu Arg 85 90 95 Lys AlaLys Arg Thr Arg Ala Val Gln Pro Leu Arg Leu Pro Ser Asn 100 105 110 LysAla Gln Val Lys Pro Gly Gln Thr Cys Ser Val Ala Gly Trp Gly 115 120 125Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu Gln Glu Val Lys 130 135140 Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp Leu Arg His Tyr 145150 155 160 Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu Ile LysLys 165 170 175 Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val Cys AsnLys Val 180 185 190 Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly MetPro Pro Arg 195 200 205 Ala Cys Thr Lys Val Ser Ser Phe Val His Trp IleLys Lys Thr Met 210 215 220 Lys Arg Tyr Gly Gly Gly Gly Ser Ala Pro MetAla Glu Gly Gly Gly 225 230 235 240 Gln Asn His His Glu Val Val Lys PheMet Asp Val Tyr Gln Arg Ser 245 250 255 Tyr Cys His Pro Ile Glu Thr LeuVal Asp Ile Phe Gln Glu Tyr Pro 260 265 270 Asp Glu Ile Glu Tyr Ile PheLys Pro Ser Cys Val Pro Leu Met Arg 275 280 285 Cys Gly Gly Cys Cys AsnAsp Glu Gly Leu Glu Cys Val Pro Thr Glu 290 295 300 Glu Ser Asn Ile ThrMet Gln Ile Met Arg Ile Lys Pro His Gln Gly 305 310 315 320 Gln His IleGly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys 325 330 335 Arg ProLys Lys Asp Arg Ala Arg Gln Glu Asn Cys Asp Lys Pro Arg 340 345 350 Arg58 1440 DNA Artificial Sequence Description of Artificial SequenceGranzyme B-scFvMEL 58 atcatcgggg gacatgaggc caagccccac tcccgcccctacatggctta tcttatgatc 60 tgggatcaga agtctctgaa gaggtgcggt ggcttcctgatacaagacga cttcgtgctg 120 acagctgctc actgttgggg aagctccata aatgtcaccttgggggccca caatatcaaa 180 gaacaggagc cgacccagca gtttatccct gtgaaaagacccatccccca tccagcctat 240 aatcctaaga acttctccaa cgacatcatg ctactgcagctggagagaaa ggccaagcgg 300 accagagctg tgcagcccct caggctacct agcaacaaggcccaggtgaa gccagggcag 360 acatgcagtg tggccggctg ggggcagacg gcccccctgggaaaacactc acacacacta 420 caagaggtga agatgacagt gcaggaagat cgaaagtgcgaatctgactt acgccattat 480 tacgacagta ccattgagtt gtgcgtgggg gacccagagattaaaaagac ttcctttaag 540 ggggactctg gaggccctct tgtgtgtaac aaggtggcccagggcattgt ctcctatgga 600 cgaaacaatg gcatgcctcc acgagcctgc accaaagtctcaagctttgt acactggata 660 aagaaaacca tgaaacgcta cggtggcggt ggctccacggacattgtgat gacccagtct 720 caaaaattca tgtccacatc agtaggagac agggtcagcgtcacctgcaa ggccagtcag 780 aatgtggata ctaatgtagc ctggtatcaa caaaaaccagggcaatctcc tgaaccactg 840 cttttctcgg catcctaccg ttacactgga gtccctgatcgcttcacagg cagtggatct 900 gggacagatt tcactctcac catcagcaat gtgcagtctgaagacttggc agagtatttc 960 tgtcagcaat ataacagcta tcctctgacg ttcggtggaggcaccaagct ggagatcaaa 1020 ggctccacca gcggcagcgg taagccaggc tccggcgaaggcagcaccaa aggcgaagtg 1080 aaggttgagg agtctggagg aggcttggtg caacctggaggatccatgaa actctcctgt 1140 gttgtctctg gattcacttt cggtaattac tggatgaactgggtccgcca gtctccagag 1200 aaggggcttg agtggattgc agaaattaga ttgaaatccaataattttgc aagatattat 1260 gcggagtctg tgaaagggag gttcaccatc tcaagagatgattccaaaag tagtgtctac 1320 ctgcaaatga tcaacctaag agctgaagat actggcatttattactgtac cagttatggt 1380 aactacgttg ggcactattt tgaccactgg ggccaaggcaccactctcac cgtctcctca 1440 59 480 PRT Artificial Sequence Description ofArtificial Sequence Granzyme B-scFvMEL 59 Ile Ile Gly Gly His Glu AlaLys Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 Tyr Leu Met Ile Trp AspGln Lys Ser Leu Lys Arg Cys Gly Gly Phe 20 25 30 Leu Ile Gln Asp Asp PheVal Leu Thr Ala Ala His Cys Trp Gly Ser 35 40 45 Ser Ile Asn Val Thr LeuGly Ala His Asn Ile Lys Glu Gln Glu Pro 50 55 60 Thr Gln Gln Phe Ile ProVal Lys Arg Pro Ile Pro His Pro Ala Tyr 65 70 75 80 Asn Pro Lys Asn PheSer Asn Asp Ile Met Leu Leu Gln Leu Glu Arg 85 90 95 Lys Ala Lys Arg ThrArg Ala Val Gln Pro Leu Arg Leu Pro Ser Asn 100 105 110 Lys Ala Gln ValLys Pro Gly Gln Thr Cys Ser Val Ala Gly Trp Gly 115 120 125 Gln Thr AlaPro Leu Gly Lys His Ser His Thr Leu Gln Glu Val Lys 130 135 140 Met ThrVal Gln Glu Asp Arg Lys Cys Glu Ser Asp Leu Arg His Tyr 145 150 155 160Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu Ile Lys Lys 165 170175 Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Lys Val 180185 190 Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly Met Pro Pro Arg195 200 205 Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile Lys Lys ThrMet 210 215 220 Lys Arg Tyr Gly Gly Gly Gly Ser Thr Asp Ile Val Met ThrGln Ser 225 230 235 240 Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg ValSer Val Thr Cys 245 250 255 Lys Ala Ser Gln Asn Val Asp Thr Asn Val AlaTrp Tyr Gln Gln Lys 260 265 270 Pro Gly Gln Ser Pro Glu Pro Leu Leu PheSer Ala Ser Tyr Arg Tyr 275 280 285 Thr Gly Val Pro Asp Arg Phe Thr GlySer Gly Ser Gly Thr Asp Phe 290 295 300 Thr Leu Thr Ile Ser Asn Val GlnSer Glu Asp Leu Ala Glu Tyr Phe 305 310 315 320 Cys Gln Gln Tyr Asn SerTyr Pro Leu Thr Phe Gly Gly Gly Thr Lys 325 330 335 Leu Glu Ile Lys GlySer Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly 340 345 350 Glu Gly Ser ThrLys Gly Glu Val Lys Val Glu Glu Ser Gly Gly Gly 355 360 365 Leu Val GlnPro Gly Gly Ser Met Lys Leu Ser Cys Val Val Ser Gly 370 375 380 Phe ThrPhe Gly Asn Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu 385 390 395 400Lys Gly Leu Glu Trp Ile Ala Glu Ile Arg Leu Lys Ser Asn Asn Phe 405 410415 Ala Arg Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 420425 430 Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Ile Asn Leu Arg Ala435 440 445 Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Ser Tyr Gly Asn Tyr ValGly 450 455 460 His Tyr Phe Asp His Trp Gly Gln Gly Thr Thr Leu Thr ValSer Ser 465 470 475 480 60 247 PRT Homo sapiens MOD_RES (55) x = q or r60 Met Gln Pro Ile Leu Leu Leu Leu Ala Phe Leu Leu Leu Pro Arg Ala 1 510 15 Asp Ala Gly Glu Ile Ile Gly Gly His Glu Ala Lys Pro His Ser Arg 2025 30 Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg 3540 45 Cys Gly Gly Phe Leu Ile Xaa Asp Asp Phe Val Leu Thr Ala Ala His 5055 60 Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys 6570 75 80 Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg Glx Ile Pro85 90 95 His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu100 105 110 Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln Pro LeuArg 115 120 125 Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr CysSer Val 130 135 140 Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His SerHis Thr Leu 145 150 155 160 Gln Glu Val Lys Met Thr Val Gln Glu Asp ArgLys Cys Glu Ser Asp 165 170 175 Leu Arg His Tyr Tyr Asp Ser Thr Ile GluLeu Cys Val Gly Asp Pro 180 185 190 Glu Ile Lys Lys Thr Ser Phe Lys GlyAsp Ser Gly Gly Pro Leu Val 195 200 205 Cys Asn Lys Val Ala Gln Gly IleVal Ser Tyr Gly Arg Asn Asn Gly 210 215 220 Met Pro Pro Arg Ala Cys ThrLys Val Ser Ser Phe Val His Trp Ile 225 230 235 240 Lys Lys Thr Met LysArg Tyr 245 61 34 PRT Homo sapiens 61 Met Lys Ser Leu Ser Leu Leu HisLeu Phe Pro Leu Pro Arg Ala Lys 1 5 10 15 Arg Glu Gln Gly Gly Asn AsnSer Ser Ser Asn Gln Gly Ser Leu Pro 20 25 30 Glu Lys

We claim:
 1. A chimeric polypeptide comprising a cell-specific targetingmoiety and a signal transduction pathway factor.
 2. A chimericpolypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme.
 3. The polypeptide of claim 2, wherein said granzyme isgranzyme B.
 4. The polypeptide of claim 3, wherein the amino acidsequence of said granzyme B is SEQ ID NO:60.
 5. The polypeptide of claim3, wherein the amino acid sequence of said granzyme B is at least 100contiguous amino acids from SEQ ID NO:60.
 6. The polypeptide of claim 3,wherein the amino acid sequence of said granzyme B is at least 75contiguous amino acids from SEQ ID NO:60.
 7. The polypeptide of claim 3,wherein the amino acid sequence of said granzyme B is at least 40contiguous amino acids from SEQ ID NO:60.
 8. The polypeptide of claim 4,wherein SEQ ID NO:60 further comprises an N-terninal extensioncomprising SEQ ID NO:61.
 9. The polypeptide of claim 4, wherein thefirst twenty amino acids are absent from SEQ ID NO:60.
 10. Thepolypeptide of claim 2, wherein said granzyme is granzyme A.
 11. Thepolypeptide of claim 10, wherein the amino acid sequence of saidgranzyme A is SEQ ID NO:25.
 12. The polypeptide of claim 10, wherein theamino acid sequence of said granzyme A is at least 100 contiguous aminoacids from SEQ ID NO:25.
 13. The polypeptide of claim 10, wherein theamino acid sequence of said granzyme A is at least 75 contiguous aminoacids from SEQ ID NO:25.
 14. The polypeptide of claim 10, wherein theamino acid sequence of said granzyme A is at least 40 contiguous aminoacids from SEQ ID NO:25.
 15. The polypeptide of claim 2, wherein saidcell-specific targeting moiety is a cytokine, an antibody, a ligand, ora hormone.
 16. The polypeptide of claim 15, wherein said ligand is VEGF.17. The polypeptide of claim 16, wherein said VEGF is vegf121.
 18. Thepolypeptide of claim 15, wherein said antibody is a single chainantibody.
 19. The polypeptide of claim 18, wherein said single chainantibody is scFvMEL.
 20. The polypeptide of claim 2, wherein saidgranzyme is granzyme B and said cell-specific targeting moiety isvegf121.
 21. The polypeptide of claim 2, wherein said granzyme isgranzyme B and said cell-specific targeting moiety is scFvMEL.
 22. Thepolypeptide of claim 2, further comprising a linker.
 23. The polypeptideof claim 22, wherein the linker comprises SEQ ID NO:50, SEQ ID NO:51, orSEQ ID NO:52.
 24. The polypeptide of claim 2, wherein the polypeptide isencoded by a recombinant polynucleotide.
 25. An expression cassettecomprising a polynucleotide encoding a chimeric polypeptide comprising acell-specific targeting moiety and an apoptosis-inducing factor, whereinsaid apoptosis-inducing factor is a granzyme, and wherein saidpolynucleotide is under control of a regulatory sequence operable in ahost cell.
 26. The expression cassette of claim 25, wherein saidgranzyme is granzyme A.
 27. The expression cassette of claim 25, whereinsaid granzyme is granzyme B.
 28. The expression cassette of claim 26,wherein said granzyme A is encoded by a polynucleotide of SEQ ID NO:26,SEQ ID NO:27, or SEQ ID NO:28.
 29. The expression cassette of claim 27,wherein said granzyme B is encoded by a polynucleotide of SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.30. The expression cassette of claim 25, wherein the cassette iscomprised in a recombinant viral vector.
 31. The expression cassette ofclaim 30, wherein the viral vector is an adenoviral vector, anadeno-associated viral vector, or a retroviral vector.
 32. A host cellcomprising an expression cassette comprising a polynucleotide encoding achimeric polypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme.
 33. The host cell of claim 32, further defined as aprokaryotic host cell.
 34. The host cell of claim 32, further defined asan eukaryotic host cell.
 35. A method of using a host cell comprising anexpression cassette comprising a polynucleotide encoding a chimericpolypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme, the method comprising culturing the host cell under conditionssuitable for the expression of the chimeric polypeptide.
 36. A method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a granzyme.
 37. The method of claim 36, whereinsaid granzyme is granzyme A or granzyme B.
 38. The method of claim 37,wherein said granzyme is granzyme B.
 39. The method of claim 36, whereinsaid cell is in vivo.
 40. The method of claim 39, wherein said cell isin a human.
 41. A method of inducing apoptosis in a cell, comprisingadministering to said cell an effective amount of a chimeric polypeptidecomprising a cell-specific targeting moiety and a granzyme, wherein saidcell-specific targeting moiety is scFvMEL and said granzyme is granzymeB.
 42. A method of inducing apoptosis in a cell, comprisingadministering to said cell an effective amount of a chimeric polypeptidecomprising a cell-specific targeting moiety and a granzyme, wherein saidcell-specific targeting moiety is vegf121 and said granzyme is granzymeB.
 43. A method of inducing apoptosis in a cell, comprisingadministering to said cell an effective amount of a chimeric polypeptidecomprising a cell-specific targeting moiety and a pro-apoptotic memberof the Bcl-2 family.
 44. The method of claim 43, wherein saidpro-apoptotic member of the Bcl-2 family is Bax or a fragment thereof.45. The method of claim 43, wherein said cell is in vivo.
 46. The methodof claim 45, wherein said cell is in a human.
 47. The method of claim44, wherein said fragment of Bax lacks at least part of a polypeptideencoded by exon 6 in a Bax polynucleotide sequence.
 48. A method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a pro-apoptotic member of the Bcl-2 family, whereinsaid cell-specific targeting moiety is scFvMEL and said pro-apoptoticmember of the Bcl-2 family is Bax or a fragment of Bax.
 49. The methodof claim 48, wherein said fragment of Bax lacks at least part of exon 6in a Bax polynucleotide sequence.
 50. A method of treating a disease inan individual, comprising the steps of administering to said individuala therapeutically effective amount of a composition comprising: a) achimeric polypeptide comprising an apoptosis-inducing moiety and acell-specific targeting moiety; and b) a pharmaceutical carrier.
 51. Themethod of claim 50, wherein said pharmaceutical carrier comprises alipid.
 52. The method of claim 50, wherein said disease is cancer,diabetes, arthritis, or inflammatory bowel disease, atherosclerosis, ordiabetic retinopathy.
 53. The method of claim 52, wherein said diseaseis cancer.
 54. The method of claim 50, wherein said apoptosis-inducingmoiety is a granzyme.
 55. The method of claim 54, wherein said granzymeis granzyme B or a fragment thereof.
 56. The method of claim 50, whereinsaid apoptosis-inducing moiety is a pro-apoptotic member of the Bcl-2family.
 57. The method of claim 56, wherein said pro-apoptotic member ofthe Bcl-2 family is Bax or a fragment thereof.
 58. The method of claim57, wherein said fragment of Bax lacks at least part of a polypeptideencoded by exon 6 in a Bax polynucleotide sequence.
 59. The method ofclaim 57, wherein said fragment of Bax lacks at least part of apolypeptide encoded by exons 4, 5, or
 6. 60. The method of claim 50,wherein said administration is intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally, byinhalation (e.g. aerosol inhalation), by injection, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in a creme, or in a lipidcomposition.
 61. The method of claim 50, further comprisingadministering to said individual an anti-inflammatory composition,chemotherapy, surgery, radiation, hormone therapy, or gene therapy.